Compositions comprising acidic extracts of mastic gum

ABSTRACT

The invention relates to compositions and formulations comprising isolated acidic fraction of mastic gum and uses thereof for treating impaired neurological functions as well as wound and tissue repair.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/677,365, filed on Aug. 15, 2017, which is a divisional of U.S.patent application Ser. No. 15/000,849, filed on Jan. 19, 2016, now U.S.Pat. No. 9,770,456 issued on Sep. 26, 2017, which is a divisional ofU.S. patent application Ser. No. 13/821,194, filed on Mar. 6, 2013, nowU.S. Pat. No. 9,271,949 issued on Mar. 1, 2016, which is a 35 U.S.C. §371 National Phase Entry Application from PCT/IL2011/000724 filed onSep. 7, 2011, which claims priority under 35 U.S.C. § 119 to U.S.Provisional Patent Application No. 61/380,339 filed on Sep. 7, 2010,which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to compositions of isolated acidic fractions frommastic gum, and uses thereof.

BACKGROUND OF THE INVENTION

The pursuit of new drug entities derived from plants and plant productsfor various therapeutic applications has its origins in antiquity andcontinues to the present. One such source is mastic, also known as gummastic or mastic gum, which is a tree resin obtained as an exudate fromPistacia lentiscus L, a member of the family Anacardiaceae. Mastic wasused in the ancient Mediterranean world for gastrointestinal disorderssuch as gastralgia, dyspepsia and peptic ulcer. Oral administration ofmastic to human patients with duodenal ulcer and to experimental ratswith induced gastric and duodenal ulcers has been disclosed to havetherapeutic effects (Al-Habbal et al (1984) Clin Exp Pharmacop Physio11(5):541-4; Said at al (1986) J Ethnopharmacol 15(3):271-8). While ithas been disclosed that mastic has in vitro bactericidal effects againstHelicobacter pylori, the etiologic agent causing peptide ulcer disease(Marone et at (2001) J Chemother 13:611-614), other reports disclosethat mastic does not exert anti-bacterial activity upon administrationto H. pylori positive human patients (Bebb et al (2003) J AntimicrobChemother 52:522-23) or to experimentally infected mice (Loughlin et at(2003) J Antimicrob Chemother 51:367-371).

Greek Patent No. GR 1,003,541 discloses antimicrobial and antifungalaction of the chios mastic oil extracted from the leaves, branches andfruit of Pistacia lentiscus var Chia.

Greek Patent No. GR 1,003,868 discloses use of a product derived fromPistacia lentiscus var. Chia as an antioxidant, as a wound healinginductor and as a cytostatic agent.

U.S. Patent Application Publication No 2005/0238740 is directed to Useof mastic and its components for the control of microbial infections.

Paraschos et al (2007), authored by some of the inventors of theaforementioned patent application, disclose preparation of a totalmastic extract without polymer (TMEWP), prepared by polar solventextraction of crude mastic and removal of the insoluble polymerpoly-1-myrcene therefrom, and acidic and neutral fractions separatedfrom TMEWP (Paraschos et al (2007) Antimicrob Agents Chemother51(2):551-559). According to the disclosure, administration of TMEWP toH. pylori infected mice over a period of 3 months resulted in a 30-foldreduction of bacterial colonization, largely attributable to aparticular compound purified from the acid fraction. The authorsindicate that TMEWP was prepared since the high percentage ofpoly-β-myrcene in crude mastic preparations, as used in previousstudies, was speculated to hinder potential in vivo activity during oraladministration. The authors further disclose that removal of thepoly-β-myrcene can produce an enhanced therapeutic moiety with anti-H.pylori activity.

EP Patent Application No. 1520585 is directed to Cancer treatment usingnatural plant products or essential oils or components from somepistacia species.

International Patent Application Publication No. WO 2005/112967 isdirected to anticancer activity of chios mastic gum.

Van der Berg et al (1998) disclose isolation and purification of thepolymer fraction of mastic using extraction and size exclusionchromatography (Van der Berg et al (1998) Tetrahedron Lett 3:2645-2648).

Barra et al (2007) disclose extraction and gas chromatographic analysisof essential oil from P. lentiscus L. (Barra et al (2007) J Agric FoodChem 55(17):7093-7098). According to the disclosure, a total of 45compounds were identified, including β-myrcene as one of the majorcompounds.

International Patent Application Publication No. WO 2010/100650 to someof the inventors of the present invention, is directed to therapeuticuses of mastic gum fractions.

International Patent Application Publication No. WO 2010/100651 to someof the inventors of the present invention, is directed to compositionsof polymeric myrcene.

International Patent Application Publication No. WO 2005/094837 isdirected to Use of masticadienoic acid as inhibitor of DNApolymerase-beta, used for treating cancers, tumors and neurodegenerativediseases.

Marner et al (1991) disclose identification of various triterpenoidsfrom gum mastic of P. lentiscus (Marner et al (1991) Phytochemistry, 30,3709-3712).

Giner-Larza et al (2002) disclose anti-inflammatory triterpenes frompistacia terebinthus galls (Planta Med (2002), 68, 311-315).

Nevertheless, there remains an unmet need for safe, versatile andeffective agents for treatment of various conditions, such as thoseassociate with impaired neurological functions and relatedneurodegenerative conditions, including, for example, Alzheimer'sdisease, stroke, and the like, as well and other conditions, such astissue regeneration, wound and tissue repair.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprising anisolated acidic fraction of mastic gum, which contains acidic compoundsthat are soluble in both polar and non-polar organic solvents. Thefraction of the invention exhibits a variety of beneficial biologicalactivities which may be exploited for various therapeutic applications.More specifically, an isolated acidic fraction of mastic gum is nowdisclosed to have activity and be useful in treating impairedneurological functions and related neurodegenerative conditions (forexample, by reversal of the neurodegenerative condition), stroke, tissueregeneration, wound and tissue repair, and the like.

According to some embodiments, the present invention further providescompositions comprising isolated compounds from the isolated acidicfraction of mastic gum, having therapeutic activity. In someembodiments, the compositions may include a plurality of isolatedcompounds selected from the individual acidic compounds found in theacidic fraction of mastic gum according to the invention. According tosome embodiments the composition includes at least three isolatedcompounds selected from masticadienoic acid, isomasticadienoic acid,masticadienolic acid, isomasticadienolic acid, 3-O-acetylmasticadienolic acid, 3-O-acetyl epimasticadienolic acid, 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid,oleanonic acid, moronic acid and 3-oxo-lup-20(29)-en-28-oic acid. Insome exemplary embodiments, the compositions include at leastmasticadienoic acid, isomasticadienoic acid and oleanonic acid. Suchcompositions unexpectedly exhibit a synergistic effect, whereby thecombination of compounds exhibit a markedly improved therapeutic effectin the treatment of impaired neurological functions andneurodegenerative disorders/conditions, as compared to the individualcompounds alone.

The mastic acidic fraction according to embodiments of the invention maybe distinguished over mastic fractions disclosed in the prior art, asits preparation involves use of both a polar solvent and a non-polarsolvent, whereas the prior art teaches use of polar solvents only.Acidic fractions prepared by using only polar solvents contain compoundsthat are not soluble in apolar solvents such as hexane, whereas thesecompounds are not present in the acidic fraction of the currentinvention. Accordingly, the fractions of the invention comprise acombination of compounds which differs from that disclosed in the priorart. Moreover, the inventors of the present invention have discoveredthat the acidic fraction of the invention unexpectedly possesses a rangeof highly unexpected therapeutic activities that are not suggested bythe prior art.

The teachings of the present invention have been exemplified with masticgum extracts prepared by a three-step extraction procedure, so as toobtain an acidic fraction that is soluble in both a polar solvent and anon-polar solvent, and wherein material from the mastic gum that issoluble in the polar solvent but remains insoluble in the non-polarsolvent is eliminated. In further embodiments, main compounds of theisolated acidic fraction have been isolated and identified. Variouscombinations of some of these compounds exhibit an unexpectedsynergistic effect in the treatment of various impaired neurologicalfunctions (such as, for example, stroke), and in the treatment ofrelated neurodegenerative disorders such as Alzheimer's disease.

Without wishing to be bound by any particular theory or mechanism ofaction, the activity of the isolated acidic fractions of mastic gumdisclosed herein renders the present invention useful for variousdisease or conditions associated with impaired neuronal conditions, suchas, for example, reformation of inter-neuronal junctions and overcomingdefective inter-neuronal communication in brain and neural tissueaffected by pathologies associated with inadequate synaptic formation.This pathology underlies many nervous system pathologies, including forexample Alzheimer's disease. It is shown that the activity of theisolated acidic fractions of mastic gum may be used for the treatment ofstroke. The invention may be further useful for promoting wound healingand rejuvenation of a large number of cells and tissues. The inventionmay also be useful for extending the life span of animals.

It is to be further understood that the biological activity of thefractions and compositions disclosed herein is inhibited by the presenceof certain compounds that are be present in acidic fractions that havebeen prepared without applying the first two extraction steps asdisclosed herein.

According to some embodiments, the present invention provides acomposition comprising an effective amount of an isolated acidicfraction of mastic gum, and a pharmaceutically acceptable carrier;wherein the fraction is characterized in that it is soluble in at leastone polar organic solvent and soluble in at least one non-polar organicsolvent, and wherein said fraction is substantially devoid of compoundswhich are soluble in said polar organic solvent but insoluble in saidnon-polar organic solvent.

In some embodiments, the composition is obtained by a process comprisingthe steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent,    -   (e) isolating a fraction soluble in said non-polar organic        solvent;    -   (f) optionally removing said non-polar organic solvent;    -   (g) dissolving the fraction obtained in step (f) in an organic        solvent;    -   (h) treating the solution obtained in step (g) with a basic        solution so as to obtain a basic fraction; and    -   (i) acidifying the basic fraction obtained in step (h) with an        acid solution;

In some embodiments, steps (d) to (f) may precede steps (a) to (c).

In some embodiments, the treatment with a basic solution (basifying) instep (h) comprises extracting the solution obtained in step (g) with oneor more suitable basic aqueous solutions; or contacting the solutionobtained in step (g) with a basic ion exchange resin.

In some embodiments, step (h) comprises contacting the solution obtainedin step (g) with a basic ion exchange resin, and thereafter removing thebasic ion exchange resin by filtration. In these embodiments, step (i)comprises treating the basic ion exchange resin with an acidic solution.

In some embodiments, the process further comprises the steps of

-   -   (j) extracting the acidified fraction obtained in step (i) with        an organic solvent; k) optionally contacting the organic        fraction obtained in step (j) with a drying agent so as to        remove remaining water;    -   (l) removing organic solvent and/or excess acid from the        fraction obtained in any of steps (i), (j) or (k); and    -   (m) dissolving the isolated fraction obtained in step (l) in a        carrier.

In some embodiments, steps (a) to (c) are carried out prior to steps (d)to (f); or steps (d) to (f) are carried out prior to steps (a) to (c).In some embodiments, (a) to (c) and/or steps (d) to (f) are repeated fora multiplicity of cycles.

In some embodiments, any of steps (c), (f) and (l) comprise removing thesolvent by a means selected from the group consisting of rotaryevaporation, application of high vacuum and a combination thereof.

In some embodiments, step (h) comprises extracting the solution obtainedin step (g) with a basic aqueous solution, and collecting the organicfraction thus obtained. In some embodiments, the process furthercomprises combining the organic fraction obtained from step (h) with afraction obtained in any of steps (i), (j) or (k).

In some embodiments, the organic fraction obtained in step (h) iscombined with a fraction obtained in any of steps (i), (j) or (k) in anamount in the range from about 0.1% to about 50% of the organic fractionobtained from step (h). In some embodiments, the amount is in the rangefrom 0.5 to 50%; or 2 to 25%; or 0.1 to 10%.

Polar organic solvents suitable for use in the invention may be selectedfrom an alcohol, an ether, an ester, an amide, an aldehyde, a ketone, anitrile, and combinations thereof.

Specific examples of suitable polar organic solvents include methanol,ethanol, propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol,t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, neopentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol,2-methyl-2-butanol, ethyleneglycol, ethyleneglycol monomethyl ether,diethyl ether, methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and combinationsthereof.

In some embodiments, the polar organic solvent is selected from methanoland ethanol or a combination thereof.

In some embodiments, the polar solvent is ethanol.

Non-polar organic solvents suitable for use in the invention may beselected from acyclic or cyclic, saturated or unsaturated aliphatichydrocarbons and aromatic hydrocarbons, each of which is optionallysubstituted by one or more halogens, and combinations thereof. In someembodiments, the non-polar organic solvent is selected from C5-C10alkanes, C5-C10 cycloalkanes, C6-C14 aromatic hydrocarbons and C7-C14perfluoroalkanes, and combinations thereof.

In some embodiments, the non-polar organic solvent is selected frompentanes, hexanes, heptanes, octanes, nonanes, decanes, cyclopentane,cyclohexane, cycloheptane, benzene, toluene, xylene, and isomers andmixtures thereof.

In some embodiments, the C5-C10 alkane is selected from the groupconsisting of pentane, hexane, heptane, octane, nonane, decane,cyclohexane, and isomers and mixtures thereof.

In some embodiments, the non-polar organic solvent is hexane.

In some embodiments, the organic solvent in step (g) and in step (j) isindependently selected from the group consisting of dialkyl ethers,alkyl-aryl ethers, diaryl ethers, esters, ketones, halogenatedhydrocarbons, C5-C14 aromatic hydrocarbons, C5-C14 perfluoroalkanes.

In some embodiments, the suitable organic solvent in step (g) and instep (i) is the same or different.

In some embodiments, the organic solvent comprises a dialkyl ether.

In some embodiments, the organic solvent is diethyl ether.

In some embodiments, the polar organic solvent comprises ethanol, thenon-polar organic solvent comprises hexane and the organic solventcomprises diethyl ether.

In some embodiments, step (h) comprises basifying with a basic aqueoussolution. In some embodiments, the basic aqueous solution is prepared bydissolving an inorganic base in water.

In some embodiments, the inorganic base is selected from the groupconsisting of sodium carbonate, sodium hydroxide, potassium carbonatepotassium hydroxide, ammonium hydroxide, sodium bicarbonate, sodiumphosphate, lithium hydroxide, lithium carbonate, and potassiumphosphate.

In some embodiments, the inorganic base is sodium carbonate. In someembodiments, the concentration of the sodium carbonate in water is inthe range from 2 to 20% w/w. In some embodiments, the concentration ofsodium carbonate is in the range from 3 to 15% w/w. In some embodiments,the concentration of sodium carbonate is about 5% w/w.

In some embodiments, the inorganic base is sodium hydroxide.

In some embodiments, the basic aqueous solution is prepared bydissolving a water-soluble organic base in water.

In some embodiments, the first inorganic base is about 5% w/w aqueoussodium carbonate, followed by about 4% w/w aqueous sodium hydroxide.

In some embodiments, the basifying in step (h) comprises contacting thesolution obtained in step (g) with a basic ion exchange resin. In someembodiments, the basic ion exchange resin comprises styrenedivinylbenzene, polyacrylic or formophenolic copolymers.

In some embodiments, basifying the solution is done to a pH of aboveabout 7.

In some embodiments, basifying the solution is done to a pH range of8-10.

In some embodiments, basifying the solution is done to a pH range of10-13.

In some embodiments, basifying the solution is done to a pH of >13.

In some embodiments, the acidic solution in step (i) comprises an acidicaqueous solution or an acidic non-aqueous solution.

In some embodiments, the acidic aqueous solution in step (i) is preparedby dissolving an inorganic acid in water or by diluting a concentratedmineral acid solution.

In some embodiments, the acidic aqueous solution is a solution ofhydrochloric acid or phosphoric acid.

In some embodiments, the acidic aqueous solution is a solution ofhydrochloric acid.

In some embodiments, the acidic aqueous solution in step (i) is preparedby dissolving an organic acid in water or by diluting a concentratedmineral acid solution.

In some embodiments, acidifying is done to a pH of below about 7. Insome embodiments, acidifying is done to a pH of below about 6. In someembodiments, acidifying is done to a pH of below about 5. In someembodiments, acidifying is done to a pH of below about 4. In someembodiments, acidifying is done to a pH of below about 3.

In some embodiments, acidification is done to a pH in the range of 1-3.

In some embodiments, the acidic non-aqueous solution in step (i) isprepared by dissolving an organic acid in a non-aqueous organic solventselected from an alcohol, an ester, an ether, an amide or mixturesthereof. In some embodiments, the non-aqueous solvent is methanol orethanol or a mixture thereof.

In some embodiments, the organic acid is selected from the groupconsisting of formic acid, acetic acid, propionic acid, citric acid,tartaric acid, methane sulphonic acid, and para-toluenesulphonic acid.

In some embodiments, the drying agent used in step (k) is selected fromthe group of sodium sulfate, magnesium sulfate, calcium sulfate, calciumchloride, magnesium chloride, potassium sulfate.

In some embodiments, the composition is substantially devoid of terpenecompounds which are soluble in said polar organic solvent and insolublein said non-polar organic solvent.

In some embodiments, the composition comprises from about 0.01 to about25% (w/w) of the isolated acidic fraction of mastic gum, based on thetotal weight of the composition. In some embodiments, the compositioncomprises from about 0.01 to about 12% (w/w) of the isolated acidicfraction of mastic gum, based on the total weight of the composition.

In some embodiments, the isolated acidic fraction comprises at least oneof: masticadienolic acid; isomasticadienoic acid; isomasticadienolicacid; 3-O-acetyl masticadienolic acid; 3-O-acetyl epimasticadienolicacid; 3-O-acetyl isomasticadienolic acid, 3-O-acetylepi-isomasticadienolic acid; oleanonic acid; moronic acid; and3-oxo-lup-20(29)-en-28-oic acid. Each possibility is a separateembodiment of the invention.

In some embodiments, the isolated acidic fraction further comprises atleast one of: oleanolic acid; ursonic acid; and ursolic acid. Eachpossibility is a separate embodiment of the invention.

In some embodiments, the isolated acidic fraction is substantiallydevoid of masticadienoic acid.

In some embodiments, the isolated acidic fraction may be substantiallydevoid of essential oils.

In some embodiments, the isolated acidic fraction comprises at least oneterpenoic acid.

In some embodiments, the isolated acidic fraction comprises at least onetriterpenoic acid.

In some embodiments, the at least one terpenoic acid comprises at leastone triterpenoic acid. In some embodiments, the at least onetriterpenoic acid is selected from the group consisting ofmasticadienolic acid; isomasticadienoic acid; isomasticadienolic acid;3-O-acetyl masticadienolic acid; 3-O-acetyl epimasticadienolic acid;3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid; oleanonic acid; moronic acid; 3-oxo-lup-20(29)-en-28-oic acid anda combination thereof. Each possibility is a separate embodiment of theinvention.

In some embodiments, the at least one terpenoic acid is in monomericform. In some embodiments, the at least one terpenoic acid is in anoligomeric form. In some embodiments, the oligomeric form is selectedfrom the group consisting of a dimer, a trimer, and a combinationthereof. Each possibility is a separate embodiment of the invention.

In some embodiments, the oligomeric form is a dimer.

In some embodiments, the oligomeric form is a trimer.

In some embodiments, the at least one triterpenoic acid is in monomericform. In some embodiments, the at least one triterpenoic acid is in anoligomeric form. In some embodiments, the oligomeric form is selectedfrom the group consisting of a dimer, a trimer, and a combinationthereof. Each possibility is a separate embodiment of the invention.

In some embodiments, the oligomeric form is a dimer.

In some embodiments, the oligomeric form is a trimer.

In some embodiments, the isolated acidic fraction comprises acombination of monomeric and dimeric triterpenoic acids. In someembodiments, the isolated acidic fraction comprises a combination ofmonomeric, dimeric and trimeric triterpenoic acids.

In some embodiments, the composition comprises at least one of:masticadienolic acid; isomasticadienoic acid; isomasticadienolic acid;3-O-acetyl masticadienolic acid; 3-O-acetyl epimasticadienolic acid;3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid; oleanonic acid; moronic acid; and 3-oxo-lup-20(29)-en-28-oic acid.Each possibility is a separate embodiment of the invention.

In some embodiments, the composition further comprises at least one of:oleanolic acid; ursonic acid; and ursolic acid. Each possibility is aseparate embodiment of the invention.

In some embodiments, the composition comprises at least one terpenoicacid. Embodiments of terpenoic acids are as hereinbefore described.

In some embodiments, the composition comprises at least one triterpenoicacid. Embodiments of triterpenoic acids are as hereinbefore described.

In some embodiments, the composition is substantially devoid ofmasticadienoic acid.

In some embodiments, the mastic gum is derived from a plant classifiedin the family Anacardiaceae. Suitable plants include those classified ina genus selected from the group consisting of Pistacia, Pinus, Picea,Juniperus, Alsies, Larix, Antirrhinum, Boswellia, Citrus and Gynura.

In some embodiments, suitable plants are selected from the genusPistacia.

In some embodiments, the species of Pistacia is selected from the groupconsisting of P. lentiscus, P. atlantica, P. palestina, P. saportae, P.terebinthus, P. vera and P. integerrima.

In some embodiments, the species of Pistacia is Pistacia lentiscus L Insome embodiments, the isolated acidic fraction is derived from a plantmaterial selected from the group consisting of resin, leaves, twigs,roots, flowers, seeds, buds, bark, nuts and roots.

In some embodiments, the isolated acidic fraction of mastic gum isobtained by a process comprising the steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent,    -   (e) isolating a fraction soluble in said non-polar organic        solvent;    -   (f) optionally removing said non-polar organic solvent;    -   (g) dissolving the fraction obtained in step (f) in an organic        solvent;    -   (h) treatment of the solution obtained in step (g) with a basic        solution so as to obtain a basic fraction;    -   (i) acidifying the basic fraction obtained in step (i) with an        acid solution;    -   (j) extracting the acidified fraction obtained in step (i with        an organic solvent;    -   (k) optionally contacting the organic fraction obtained in        step (k) with a drying agent so as to remove remaining water;    -   (l) removing organic solvent and/or excess acid from the        fraction obtained in any of steps (i), (j) or (k); and    -   (m) dissolving the isolated fraction obtained in step (l) in a        carrier.

In additional embodiments, the invention provides a pharmaceuticalcomposition comprising at least one triterpenoic acid; and apharmaceutically acceptable carrier. In some embodiments, the at leastone triterpenoic acid is selected from the group consisting ofmasticadienolic acid; isomasticadienoic acid; isomasticadienolic acid;3-O-acetyl masticadienolic acid; 3-O-acetyl epimasticadienolic acid;3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid; Masticadienoic acid, oleanonic acid; moronic acid;3-oxo-lup-20(29)-en-28-oic acid and a combination thereof. Eachpossibility is a separate embodiment of the invention.

According to some embodiments, the current invention provides apharmaceutical composition consisting essentially of isomasticadienoicacid and masticadienoic acid as the pharmaceutically active ingredients;and a pharmaceutically acceptable carrier. The combined presence of bothcompounds results in an enhanced/synergistic effect with respect to thecomposition's efficacy in the treatment of impaired neurologicalfunction (and hence treatment of neurodegenerative disorders/conditions)when compared with the efficacy of the individual compounds.

Either one of isomasticdienoic acid and masticadienoic acid may beisolated from a natural source such as mastic gum, or may be the productof a chemical synthesis.

In some embodiments, the ratio between the isomasticdienoic acid andmasticadienoic acid is about 1:1 w/w.

According to some embodiments, the current invention provides apharmaceutical composition consisting essentially of oleanonic acid,isomasticadienoic acid and masticadienoic acid as the solepharmaceutically active ingredients; and a pharmaceutically acceptablecarrier. The combined presence of all three compounds results in a clearsynergistic effect with respect to composition's efficacy in thetreatment of impaired neurological function and treatment ofneurodegenerative disorders when compared with the efficacy of theindividual compounds or a mixture of only two of these three compounds.

Any one of oleanonic acid, isomasticadienoic acid and masticadienoicacid may be either isolated from a natural source, such as mastic gum,or may be the product of a chemical synthesis.

In some embodiments, the ratio between the isomasticdienoic acid andmasticadienoic acid and oleanonic acid is about 1:1:1 w/w/w.

In some embodiments, the at least one triterpenoic acid is a monomer. Insome embodiments, the composition comprises monomers of oleanonic acidand moronic acid. In some embodiments, the monomers of oleanonic acidand moronic acid are the products of chemical synthesis reactions.

In some embodiments, the at least one triterpenoic acid comprises anoligomeric form. In some embodiments, the oligomeric form is selectedfrom the group consisting of a dimer, a trimer, and a combinationthereof. Each possibility is a separate embodiment of the invention. Insome embodiments, the oligomeric form is a dimer.

In some embodiments, the at least one triterpenoic acid is the productof a chemical synthesis.

In some embodiments, the at least one triterpenoic acid comprising anoligomeric form is the product of a chemical synthesis. In someembodiments, the at least on triterpenoic acid is a dimeric form and isthe product of a chemical synthesis.

In some embodiments, the at least one triterpenoic acid is derived froma natural source, in particular a plant source.

In some embodiments, the composition comprises a combination ofdifferent triterpenoic acids, wherein at least one triterpenoic acid isthe product of a chemical synthesis and at least one other triterpenoicacid is derived from a plant source.

Natural sources include plants classified in the family Anacardiaceae.Suitable plants include those classified in a genus selected from thegroup consisting of Pistacia, Pinus, Picea, Juniperus, Alsies, Larix,Antirrhinum, Boswellia, Citrus and Gynura.

In some embodiments, suitable plants are selected from the genusPistacia.

In some embodiments, the species of Pistacia is selected from the groupconsisting of P. lentiscus, P. atlantica, P. palestina, P. saportae, P.terebinthus, P. vera and P. integerrima.

In some embodiments, the species of Pistacia is Pistacia lentiscus L.

In some embodiments, the natural source is a plant material selectedfrom the group consisting of resin, leaves, twigs, roots, flowers,seeds, buds, bark, nuts and roots.

In some embodiments, the natural source is a plant classified in a genusselected from the group consisting of Ocimum, Laurus and Lavendula.

In some embodiments, the pharmaceutically acceptable carrier comprises ahydrophobic carrier. In some embodiments, the hydrophobic carriercomprises at least one oil. In some embodiments, the oil is selectedfrom the group consisting of a mineral oil, a vegetable oil andcombinations thereof. In some embodiments, the vegetable oil is selectedfrom the group consisting of cottonseed oil, olive oil, almond oil,canola oil, coconut oil, corn oil, grape seed oil, peanut oil, saffronoil, sesame oil, soybean oil, and combinations thereof. In someembodiments, the mineral oil is light mineral oil. In some embodiments,the hydrophobic carrier comprises at least one wax. In some embodiments,the hydrophobic carrier comprises a combination of at least one oil andat least one wax.

In various embodiments, a composition according to the invention is in aform suitable for administration by a route selected from the groupconsisting of parenteral, transdermal, oral and topical.

In various embodiments, a composition according to the invention is in aform suitable for topical administration.

In various embodiments, a composition according to the invention is in aform suitable for oral administration.

In various embodiments, a composition according to the invention is in aform suitable for parenteral administration.

In some embodiments, the composition is in a form suitable foradministration by injection. In various embodiments, the composition isa parenteral formulation for administration by a route selected from thegroup consisting of subcutaneous, intravenous, intramuscular,intradermal, intraperitoneal, intraarterial, intracerebral,intracerebroventricular, intraosseus and intrathecal.

In some embodiments, the composition is a parenteral formulation foradministration by subcutaneous route.

In various embodiments, the composition is a formulated foradministration by a route selected from the group consisting of dermal,vaginal, rectal, inhalation, intranasal, ocular, auricular and buccal.

In some embodiments, the composition is in a form suitable for cosmeticor dermatologic administration.

In some embodiments, the pharmaceutical composition is in a formselected from the group consisting of a capsule, a tablet, a liposome, asuppository, a suspension, an ointment, a cream, a lotion, a solution,an emulsion, a film, a cement, a powder, a glue, an aerosol and a spray.In some embodiments, the capsule is selected from the group consistingof a hard gelatin capsule and a soft gelatin capsule. In someembodiments, the emulsion is a nanoemulsion or a microemulsion.

In some embodiments, the formulation comprises at least one of aninclusion complex, a nanoemulsion, a microemulsion, a powder, a lipidraft, a lipid microparticle, a dendrimer and a liposome. In someembodiments, the inclusion complex comprises at least one cyclodextrin.In some embodiments, the at least one cyclodextrin compriseshydroxypropyl-j-cyclodextrin. In some embodiments, the nanoemulsioncomprises droplets having average particle size of less than 800 nm. Insome embodiments, the droplets have average particle size of less than500 nm. In some embodiments, the droplets have average particle size ofless than 200 nm. In some embodiments, the powder is a spray driedpowder. In some embodiments, the liposome comprises a multilamellarvesicle. In some embodiments, the microemulsion comprises a non-ionicsurfactant. In some embodiments, the non-ionic surfactant is selectedfrom the group consisting of a polyoxyl castor oil, a polyoxyethylenesorbitan fatty acid ester (polysorbates), a poloxamer, a vitamin Ederivative, a polyoxyethylene alkyl ether, a polyoxyethylene sterate, orsaturated polyglycolyzed glyceride or combinations thereof.

In some embodiments, the composition is disposed on the article ofmanufacture in the form of a coating. In some embodiments, the articleof manufacture comprises a vessel, wherein the composition is disposedwithin the vessel. In some embodiments, the article of manufacture isselected from the group consisting of a fabric article, a diaper, awound dressing, a medical device, a needle or plurality of needles, amicroneedle or plurality of microneedles, an injection device and aspray dispenser. In some embodiments, the article of manufacturecomprises a plurality of microneedles. In some embodiments, the medicaldevice is selected from the group consisting of a prosthetic, anartificial organ or component thereof, a valve, a catheter, a tube, astent, an artificial membrane, a pacemaker, a sensor, an endoscope, animaging device, a pump, a wire and an implant. In some embodiments, theimplant is selected from the group consisting of a cardiac implant, acochlear implant, a corneal implant, a cranial implant, a dentalimplant, a maxillofacial implant, an organ implant, an orthopedicimplant, a vascular implant, an intraarticular implant and a breastimplant.

In some embodiments, the composition is suitable for administration by ameans selected from the group consisting of electroporation, sonication,radio frequency, pressurized spray and combinations thereof.

In some embodiments, the composition is used for treating impairedneurological function. In some embodiments, the impaired neurologicalfunction comprises a decrease in a function selected from the groupconsisting of cognitive function, sensory function, motor function andcombinations thereof. In some embodiments, the impaired neurologicalfunction is associated with a condition or disease. In some embodiments,the condition or disease is selected from the group consisting ofAlzheimer's disease, amyotrophic lateral sclerosis (ALS), multiplesclerosis, Parkinson's disease, vascular dementia and senile dementia.In some embodiments, the condition is trauma or stroke.

In some embodiments, the condition or disease is a psychiatric disorder,such as schizophrenia, bipolar disorder or depression.

In some embodiments, the condition or disease is selected from obesity,anorexia, cachexia, an infection and an immunological disorder.

In some embodiments, the impaired neurological function is due toexposure to a drug, such as an anesthetic.

In some embodiments, the condition or disease is selected from the groupconsisting of vascular dementia, senile dementia, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson'sdisease and stroke.

In some embodiments, the condition is stroke.

In some embodiments, the condition is trauma.

In some embodiments, the condition or disease is selected from the groupconsisting of vascular dementia, senile dementia, Alzheimer's disease,amyotrophic lateral sclerosis (ALS) and multiple sclerosis.

In some embodiments, the condition or disease is selected from the groupconsisting of vascular dementia, senile dementia, Alzheimer's diseaseand amyotrophic lateral sclerosis (ALS).

In some embodiments, the condition or disease is selected from the groupconsisting of vascular dementia, senile dementia and Alzheimer'sdisease.

In some embodiments, the disease is Alzheimer's disease.

In some embodiments, the disease is amyotrophic lateral sclerosis (ALS).

In some embodiments, the composition is for treating a skin wounds,including for example, a venous leg ulcer, a pressure ulcer, a diabeticfoot ulcer, a burn, an amputation wound, a decubitus ulcer (bed sore), asplit-skin donor graft, a skin graft donor site, a medical deviceimplantation site, a bite wound, a frostbite wound, a puncture wound, ashrapnel wound, a dermabrasion, a contusion, an infection, a wound and asurgical wound.

In some embodiments, the composition is for inducing or promoting tissuerepair. As used herein, tissue repair encompasses induction andpromotion of tissue regeneration, including of neural tissues.

In some embodiments, the composition is for inducing or promoting tissuerepair following an injury or insult. In some embodiments, the injury orinsult is selected from the group consisting of a myocardial infarction,a pulmonary embolism, a cerebral infarction, peripheral artery occlusivedisease, a hernia, a splenic infarction, a venous ulcer, an axotomy, aretinal detachment, an infection and a surgical procedure.

In some embodiments, the composition is used for inducing or promotinglife span extension in animals. In some embodiments, the animals areselected from the group of humans, non-human mammals, birds and fish.

Other objects, features and advantages of the present invention willbecome clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reversed phase HPLC chromatogram of an isolated acidicfraction according to the invention. The chromatogram was obtained usinga UV-VIS detector at wavelength 205 nm detection.

FIG. 2A shows an LC-MS chromatogram of an isolated acidic fractionaccording to the invention, indicative of a mixture of triterpenoic aciddimers

FIG. 2B shows the mass spectra of the main peaks of the chromatogram ofFIG. 2A.

FIG. 2C shows an enlarged mass spectrum of the peak at retention time6.09 min, shown in FIG. 2A.

FIG. 2D shows an enlarged mass spectrum of the peak at retention time6.17 min, shown in FIG. 2A.

FIG. 3A shows a TOF mass spectrum of an isolated acidic fraction ofmastic gum, indicative of a mixture of monomeric and dimeric triterpenicacids.

FIG. 3B is the peak list for FIG. 3A.

FIG. 4 shows that preparative chromatogram of the isolated acidicfraction as prepared according to Example 1. The peak numberingcorresponds with the peak numbering of the analytical chromatogram ofFIG. 1.

FIG. 5A shows the ¹H-NMR spectrum of moronic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 5B shows the ¹³C-NMR spectrum of moronic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 6A shows the ¹H-NMR spectrum of oleanonic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 6B shows the ¹³C-NMR spectrum of oleanonic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 7A shows the ¹H-NMR spectrum of masticadienoic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 7B shows the ¹³C-NMR spectrum of masticadienoic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 8A shows the ¹H-NMR spectrum of isomasticadienoic acid, isolated bypreparative HPLC from the acidic fraction prepared according to Example1.

FIG. 8B shows the ¹³C-NMR spectrum of isomasticadienoic acid, isolatedby preparative HPLC from the acidic fraction prepared according toExample 1.

FIG. 9A shows the ¹H-NMR spectrum of 3-(O-Acetyl)-masticadienolic acid,isolated by preparative HPLC from the acidic fraction prepared accordingto Example 1.

FIG. 9B shows the ¹³C-NMR spectrum of 3-(O-Acetyl)-masticadienolic acid,isolated by preparative HPLC from the acidic fraction prepared accordingto Example 1.

FIG. 10A shows the ¹H-NMR spectrum of 3-(O-Acetyl)-isomasticadienolicacid, isolated by preparative HPLC from the acidic fraction preparedaccording to Example 1.

FIG. 10B shows the ¹³C-NMR spectrum of 3-(O-Acetyl)-isomasticadienolicacid, isolated by preparative HPLC from the acidic fraction preparedaccording to Example 1.

FIG. 11A shows the effect of the acidic fraction prepared according tothe teaching of WO 2003/097212 on ARPE-19 cells.

FIG. 11B shows the effect of the acidic fraction prepared according toExample 2 of the current invention on ARPE-19 cells.

FIG. 11C shows the effect of the cottonseed oil vehicle on ARPE-19cells.

FIG. 12A shows the effect of the hexane-insoluble fraction isolated fromthe acidic fraction prepared according to the teaching of WO2003/097212on ARPE-19 cells.

FIG. 12B shows the effect of 1% (w/w) ethanolic solution of the isolatedacidic fraction prepared according Example 1 of the current invention onARPE-19 cells.

FIG. 12C shows the effect of the ethanol vehicle on ARPE-19 cells.

FIGS. 13A-C shows pictograms of cells treated with RPh-Ac (top panel) ornon-treated cells (vehicle, bottom panel) and stained with antibodies(Ab.) to: Synaptophysin (FIG. 13A); phophorylated Akt (pAk^(Ser473))(FIG. 13B); to phopshorylated GSK3-beta^(Ser9) (FIG. 13C).

FIG. 14A shows graphs depicting the results of the rat MCAO model withrespect to removal time of adhesive tape from the contralateral paw fordifferent compositions of the current invention.

FIG. 14B shows graphs depicting the baseline fold change by thecompositions: Isomasticadienoic acid (IMDA), or a combination ofIsomasticadienoic acid and Masticadienoic acid (IMDA+MDA); orcombination of Oleanonic acid, Isomasticadienoic acid and Masticadienoic(OA+MDA+IMDA), on the removal of adhesive tape from the contralateralpaw.

FIG. 15 shows pictographs of rats demonstrating the effect of cottonseedvehicle (left hand panel), RPh-Ac (middle panel) and the combination ofOA+MDA+IMDA (right hand panel) on the healing of surgical wounds in ratsfrom the MCAo model of Example 5.

DETAILED DESCRIPTION OF THE INVENTION

The inventors of the present invention have surprisingly found thatisolated acidic fractions of mastic gum show high activity inameliorating or reversing impaired neurological function, strokerecovery, healing of skin wounds, promoting tissue repair, and inpromoting life span extension in animals.

It is herein disclosed for the first time that owing to its variousactivities in stimulating and inducing cell regeneration, the isolatedacidic fraction of mastic gum, as well as combinations of compoundsisolated therefrom, as described herein may be employed as an activeingredient in a pharmaceutical composition for a number of therapeuticindications.

Advantageously, the compositions of the invention may be used in methodsof treating impaired neurological function and skin conditions. Uponcontact with cells of both human and non-human subjects, the compositioninduces cell differentiation in a wide array of tissues, cellcompartments and cell lineages, including skin, endothelium, mucousmembranes, bones, tendons and cartilage. In addition, the celldifferentiation activity of the pharmaceutical composition may beexploited for promoting in vivo incorporation of medical devices,implants and organ transplants. Furthermore, the pharmaceuticalcomposition may be used to promote life span extension in animals.

Definitions

As used herein, the terms “mastic”, “mastic resin”, “gum mastic” and“mastic gum”, are used interchangeably to refer to a tree resin (alsoknown as an oleoresin) obtained as an exudate from any tree classifiedin the family Anacardiaceae. Trees in the genus Pistacia, most notablyPistacia lentiscus L., and in particular the cultivar P. lentiscus L.cv. Chia (cultivated on the Greek island of Chios), are known for theirhigh yield of mastic. Other varieties include P. lentiscus L. var.emarginata Engl., and P. lentiscus L. var. latifolia Coss. Additionalspecies of Pistacia include for example, P. atlantica, P. palestina, P.saportae, P. terebinthus, P. vera and P. integerrima.

As used herein, the term “isolated acidic fraction of mastic gum” refersto a fraction obtained following extraction of gum mastic with at leastone polar and at least one non-polar organic solvent, followed by anacid-base extraction of a solution of the thus obtained material andisolation of the resulting acidic fraction. The isolated acidic fractionof the invention is soluble both in polar and non-polar organicsolvents.

As used herein the term “plurality” refers to more than one, preferablymore than two. As used herein the term “synergistic” means more thanadditive.

As used herein, the term “acid-base extraction” refers to a procedure inwhich an organic solvent solution containing organic acidic and organicnon-acidic components is treated/extracted with one or more basicaqueous solution(s). As a result of this, the organic acidic componentsare deprotonated and thus converted into their corresponding ionic saltforms and as a result will dissolve in the said basic aqueous solution.The non-acidic organic components will stay behind in the originalorganic solution. Subsequently, the basic aqueous solution containingthe salt forms of the acidic components is acidified, resulting in thereformation of the protonated acid forms of the organic acidiccomponents. These protonated acid forms (acidic fraction) can be removedfrom the acidified aqueous solution in several ways depending on theproperties of the acidic compounds. One option for removing the acidicfraction from the acidified solution is by extraction into a suitableorganic solvent. Example 1 is a non-limiting example of an acid-baseextraction as described above.

Depending on the solubility of the acidic compounds in the acidifiedaqueous solution, the acidic fraction may be isolated via filtration ofthe acidified aqueous solution.

Instead of using a basic aqueous solution for the acid-base extraction,basic forms of ion-exchange resins can be used as well. In these cases,the acidic organic components (acidic fraction) are captured in theirdeprotonated anionic form by the resin. The resin is subsequentlyremoved from the initial solution, leaving non-acidic components behind.The acidic components (acidic fraction) are subsequently released fromthe resin by treatment of the resin with a suitable acidic solution.

The use of ion-exchange resins for acid-base extractions is especiallysuitable for process scale up and can be used for the development of(semi)continuous extraction processes.

Examples of the above acid-base extractions and other variations can befound in many textbooks and other publications, and are consideredcommon knowledge to those skilled in the art. An example of a usefultextbook is “Vogel's Textbook of Practical Organic Chemistry”, 5^(th)Edition, 1989, (p. 162-163).

As used herein, the term “degree of purity” refers to the content of aspecified chemical compound in a preparation, expressed as a percentageon a weight per weight basis of the specified chemical compound relativeto other chemical compounds in the preparation.

As used herein, “terpene compounds” refers to isoprene-containinghydrocarbons, having isoprene units (CH₂═C(CH₃)—CH═CH₂) in ahead-to-tail orientation. Terpene hydrocarbons in general, have themolecular formula (C₅H₈)_(n), and include hemiterpenes, (C5),monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes(C30), and tetraterpenes (C40) which respectively have 1, 2, 3, 4, 6 and8 isoprene units. Terpenes may be further classified as acyclic orcyclic.

As used herein, “terpenoids” and “terpenoid compounds” interchangeablyrefer to terpene-related compounds which contain oxygen in addition toisoprene units, and thus include alcohols, aldehydes and ketones.Terpenoids are subdivided according to the number of carbon atoms in amanner similar to terpene and thus include hemiterpenoids, (C5),monoterpenoids (C10), sesquiterpenoids (C15), diterpenoids (C20),triterpenoids (C30), and tetraterpenoids (C40) which respectively have1, 2, 3, 4, 6 and 8 isoprene units. The skeleton of terpenoids maydiffer from strict additivity of isoprene units by the loss or shift ofa fragment, generally a methyl group. Examples of monoterpenoids includecamphor, eugenol and borneol. Examples of diterpenoids include phytoland taxol. Examples of triterpenoids include squalene and lanosterol.

As used herein, “terpenoic acids” refer to terpenoid compoundscontaining at least one carboxylic acid group. The terpenoic acids mayadditionally contain one or more other oxygen-containing functionalgroups comprising hydroxyl, keto, aldehyde, ether (cyclic andnon-cyclic) and ester (cyclic and non-cyclic) groups.

As used herein, “triterpenoic acids” refer to triterpenoid compoundscontaining at least one carboxylic acid group. The triterpenoic acidsmay additionally contain one or more other oxygen-containing functionalgroups comprising hydroxyl, keto, aldehyde, ether (cyclic andnon-cyclic) and ester (cyclic and non-cyclic) groups.

As used herein, “an oligomeric form of a terpenoic acid” refers to anoligomeric terpenoid acid in which the monomeric units are either of thesame terpenoic acid or of different terpenoic acids, and are joined inany possible arrangements, and are connected one to another through anypossible bond or functional group, such as a C—C bond, an ester group oran ether group.

As used herein, “an oligomeric form of a triterpenoic acid” refers to anoligomeric triterpenoid acid in which the monomeric units are either ofthe same triterpenoic acid or of different triterpenoic acids, and arejoined in any possible arrangements, and are connected one to anotherthrough any possible bond or functional group, such as a C—C bond, anester group or an ether group.

As used herein, the terms “masticadienoic acid”, “masticadienonic acid”,“masticadienoic” and “masticadienonic acid” may interchangeably be used.

As used herein, the terms “isomasticadienoic acid”, “isomasticadienonicacid”, “isomasticadienoic” and “isomasticadienonic” may interchangeablybe used.

As used herein, “substantially devoid” means that a preparation orpharmaceutical composition according to the invention that generallycontains less than about 5% of the stated substance. For example, lessthan about 3%, less than 1%, less than 0.5%, less than 0.1%.

As used herein, “therapeutically effective amount” refers to that amountof a pharmaceutical ingredient which substantially induces, promotes orresults in a desired therapeutic effect.

As used herein, “pharmaceutically acceptable carrier” refers to adiluent or vehicle which is used to enhance the delivery and/orpharmacokinetic properties of a pharmaceutical ingredient with which itis formulated, but has no therapeutic effect of its own, nor does itinduce or cause any undesirable or untoward effect or adverse reactionin the subject.

As used herein, “pharmaceutically acceptable hydrophobic carrier” refersto a hydrophobic non-polar diluent or vehicle in which a mastic fractionis dissolved or suspended.

As used herein, “cell differentiation” refers to the process in which aless specialized cell becomes a more specialized cell. Celldifferentiation may be established on the basis of changes in any of anumber of cellular characteristics, including but not limited to size,shape, organelle appearance, membrane potential, metabolic activity, andresponsiveness to signals. A particular “grade” may be given to a celltype to describe the extent of differentiation.

As used herein, “impaired neurological function” refers to a decline ordecrease in at least one of sensory, cognitive or motor function, ascompared to a previous level of function or activity, and/or as comparedto non-impaired individuals matched according to accepted criteria.

As used herein the term “about” in reference to a numerical value statedherein is to be understood as the stated value +/−10%.

Triterpenoic Acids and Isolated Acidic Fractions Comprising TerpenoicAcids

In some embodiments, the present invention provides compositionscomprising specific combinations of terpenoic acids, such as that foundin isolated acidic fractions of mastic gum. In some embodiments, thepresent invention provides compositions consisting of specifictriterpenoic acids compounds, these compositions are shown to have anunexpected synergetic effect compared to the same individualtriterpenoic acids compounds in the treatment of neurological disorders.The triterpenoic acid compounds may be from a plant source, inparticular mastic gum, or may be the products of chemical synthesisreactions. In some cases, the compositions may correspond tocombinations of compounds, in which some are chemically synthesized andsome are derived from plant sources.

Plant species useful for obtaining the compositions of the inventioninclude without limitation, those of the genera Pistacia, Pinus, Picea,Juniperus, Alsies, Larix, Ocimum, Laurus and Lavendula.

Useful species of Pistacia include without limitation, P. lentiscus, P.atlantica, P. palestina, P. saportae, P. terebinthus, P. vera and P.Integerrima.

Commercial preparations of mastic are available for example, from theChios Gum Mastic Growers Association, or from G. Baldwin & Co., U.K.

Analytical methods for determining the precise chemical composition ofthe obtained isolated acidic fraction of mastic gum include nuclearmagnetic resonance (for example ¹HNMR and ¹³CNMR), viscometry, variousmass spectrometry methods (for example MALDI-TOF), HPLC, combinationmethods such as Liquid Chromatography-Mass spectrometry (LC-MS), UV-VISspectrometry, IR and FT-IR spectrometry and other methods as are knownin the art.

The method used for obtaining isolated acidic fractions of mastic gumcan be described as follows. By way of a general description, collectedplant material, for example mastic gum, is combined in a suitable vesselwith a suitable solvent, usually a polar solvent. Suitable polarsolvents include for example, alcohols, ethers, esters, amides,aldehydes, ketones, nitriles and combinations thereof.

Particular examples of polar organic solvents are methanol, ethanol,propanol, isopropanol, 1-butanol, 2-butanol, sec-butanol, t-butanol,1-pentanol, 2-pentanol, 3-pentanol, neopentanol, 3-methyl-1-butanol,2-methyl-1-butanol, 3-methyl-2-butanol, 2-methyl-2-butanol,ethyleneglycol, ethyleneglycol monomethyl ether, diethyl ether,methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and combinationsthereof.

The mastic gum and the solvent are preferably combined such that thesolvent is in large excess, for example 10:1 or 20:1. The mixture may beperiodically or continuously agitated over a period ranging from a fewminutes to a number of hours. The solvent may be decanted without anytreatment, or optionally the mixture may be first subjected to low speedcentrifugation, for example at 100 to 2000 rpm, as is known in the art.The insoluble material is recovered from the extract and a fresh aliquotof solvent is added to the insoluble material, such that the extractionand dissolution process is repeated for a number of cycles, in order toobtain as much as possible of the polar solvent soluble compounds. Afterthe final dissolution step, the extracts containing polar solventsoluble material are combined and the polar solvent is evaporated (forexample by using a rotary evaporation as is known in the art), so as toyield polar solvent soluble material, which may be referred to as acrude, or “first step” extract.

The first step extract material is combined with a non-polar organicsolvent and extracted by shaking over a period of 1 to 2 hours. Suitablenon-polar solvents include acyclic or cyclic, saturated or unsaturatedaliphatic hydrocarbons and aromatic hydrocarbons, for example, C5-C10alkanes, C5-C10 cycloalkanes, C6-C14 aromatic hydrocarbons, andcombinations thereof. Each of the foregoing may be optionallysubstituted by one or more halogens, for example, C7-C14perfluoroalkanes. Particular examples of non-polar organic solvents arepentanes, hexanes, heptanes, octanes, nonanes, decanes, cyclopentane,cyclohexane, cycloheptane, benzene, toluene, xylene, and isomers andmixtures thereof. Material remaining insoluble or precipitating in thepresence of the non-polar solvent is removed and discarded. Thenon-polar solvent-soluble fraction is then obtained by evaporating thenon-polar solvent (for example by rotary evaporation). This fraction maybe referred to as purified or “two step” extract, corresponding to anisolated fraction of mastic gum which is characterized by the fact thatit is soluble in both a polar solvent and a non-polar solvent, whilematerials which are soluble in the polar solvent but insoluble in thenon-polar solvent, have been removed.

The second step extract material is subsequently dissolved in an organicsolvent and this solution is extracted repeatedly (e.g. four times) witha basic aqueous solution. A second extraction with a different basicaqueous solution may be performed. The basic fraction thus obtained isacidified with a dilute aqueous acid solution to acidic pH. Theacidified aqueous solution is extracted several times with an organicsolvent. The thus obtained combined organic solvent extracts (alsoreferred to as “three step extract”) are treated with a drying agent.This isolated acidic fraction of mastic gum is then obtained byevaporating the organic solvent (for example by rotary evaporation).This fraction is referred to as the isolated acidic fraction of masticgum. Additional intermediate steps of drying and/or solvent removal maybe carried our between other steps, as is known in the art. Alternately,the second step extract material may be combined with a basic ionexchange resin e.g. Amberlyst® A26. The isolated ion-exchange resin istreated with an non-aqueous acidic solution in order to liberate theacidic fraction from the resin. The isolated acidic fraction is thenobtained by evaporating the non-aqueous solvent and any excess acid.

The feature that distinguishes the isolated acidic fractions of theinvention over prior art extracts of mastic gum is that certain acidiccompounds have been removed in the first two steps of the procedurewhich would otherwise end up in the final acidic fraction. According tothe teachings of the present invention, the acidic compounds removedduring the first two steps of the isolation procedure have a detrimentaleffect on the beneficial biological activities of the isolated acidicfractions disclosed herein.

The three step extract may be dried further, for example by high vacuumtreatment (for example <0.01 mbar for up to several days) to removeresidual solvent and other volatile material, weighed and combined witha non-polar organic solvent or other carrier to effect its dissolution.

In some embodiments, the isolated fraction of the invention may beobtained by a process comprising the steps of:

-   -   (a) treating mastic gum with a polar organic solvent;    -   (b) isolating a fraction soluble in said polar organic solvent;    -   (c) optionally removing said polar organic solvent;    -   (d) treating the soluble fraction obtained in step (b) or (c)        with a non-polar organic solvent,    -   (e) isolating a fraction soluble in said non-polar organic        solvent;    -   (f) optionally removing said non-polar organic solvent;    -   (g) dissolving the fraction obtained in step (f) in an organic        solvent;    -   (h) treating the solution obtained in step (g) with a basic        solution so as to obtain a basic fraction; and    -   (i) acidifying the basic fraction obtained in step (h) with an        acid solution.

In some embodiments, the treatment with a basic solution (basifying) instep (h) comprises extracting the solution obtained in step (g) with oneor more suitable basic aqueous solution; or contacting the solutionobtained in step (g) with a basic ion exchange resin. In case of a basicion exchange resin, the resin may subsequently be treated with an acidicsolution in order to release the captured acidic fraction. The isolatedacidic fraction is than obtained by removal of any volatiles using, forexample, application of vacuum.

In some embodiments, step (h) comprises contacting the solution obtainedin step (g) with a basic ion exchange resin, and thereafter removing thebasic ion exchange resin by filtration. The basic ion exchange resin maybe subsequently treated with an acidic solution in order to liberate thecaptured acidic fraction. The isolated acidic fraction is than obtainedby removal of any volatiles using e.g. application of vacuum.

In some embodiments, the process further comprises the steps of

-   -   (j) extracting the acidified fraction obtained in step (i) with        an organic solvent; k) optionally contacting the organic        fraction obtained in step (j) with a drying agent so as to        remove remaining water;    -   (l) removing organic solvent and/or excess acid from the        fraction obtained in any of steps (i), (j) or (k); and    -   (m) dissolving the isolated fraction obtained in step (l) in a        carrier.

The process may further comprise removing the solvent after any of steps(c), (f) or (l). Solvent removal may be carried out by any means knownin the art, for example rotary evaporation, application of high vacuumand a combination thereof. In some embodiments, steps (a) to (c) arecarried out prior to steps (d) to (f) or vice versa. In someembodiments, the polar organic solvent comprises ethanol, the non-polarorganic solvent comprises hexane and the organic solvent used for theacid-base extraction comprises diethyl ether. As is readily understoodby one of skill in the art, steps (a) to (c) and steps (d) to (f) mayeach be independently carried out for a number of cycles to optimize theextraction process and degree of purification of the product.

In some embodiments, step (h) comprises extracting the solution obtainedin step (g) with a basic aqueous solution, and collecting the organicfraction obtained therefrom. In some embodiments, the process mayfurther comprise combining the organic fraction obtained from step (h)with a fraction obtained in any of steps (i), (j) or (k).

In some embodiments, the organic fraction obtained in step (h) iscombined with a fraction obtained in any of steps (i), (j) or (k) in anamount in the range from about 0.1 to 50% of the organic fractionobtained from step (h). In some embodiments, the amount is in the rangefrom about 0.5-50%; or 2 to 25%; or 0.1 to 10%.

The isolated acidic fraction may comprise at least one terpenoic acid,such as a combination of various triterpenoic acid combinations.Triterpenoic acids include for example, masticadienolic acid;isomasticadienoic acid; isomasticadienolic acid; 3-O-acetylmasticadienolic acid; 3-O-acetyl epi-masticadienolic acid; 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid;oleanonic acid; oleanolic acid; ursonic acid; ursolic acid; moronicacid; and 3-oxo-lup-20(29)-en-28-oic acid.

In some embodiments, the isolated acidic fraction may comprise at leasttwo terpenoic acid compounds, selected from, for example,masticadienolic acid; isomasticadienoic acid; isomasticadienolic acid;3-O-acetyl masticadienolic acid; 3-O-acetyl epimasticadienolic acid;3-O-acetyl isomasticadienolic acid, 3-O-acetyl epi-isomasticadienolicacid; oleanonic acid; oleanolic acid; ursonic acid; ursolic acid;moronic acid; and 3-oxo-lup-20(29)-en-28-oic acid.

In some embodiments, the isolated acidic fraction may comprise at leastthree terpenoic acid compounds, selected from, masticadienolic acid;isomasticadienoic acid; isomasticadienolic acid; 3-O-acetylmasticadienolic acid; 3-O-acetyl epimasticadienolic acid; 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid;oleanonic acid; oleanolic acid; ursonic acid; ursolic acid; moronicacid; and 3-oxo-lup-20(29)-en-28-oic acid.

In some embodiments, the isolated acidic fraction may comprise at leasttwo terpenoic acid compounds, selected from, masticadienolic acid,isomasticadienoic acid, and oleanonic acid.

In some embodiments, the isolated acidic fraction may be substantiallydevoid of particular triterpenoic acids, such as, for example,masticadienoic acid, or moronic acid. Each possibility is a separateembodiment.

In some embodiments, the isolated acidic fraction may be substantiallydevoid of essential oils.

Furthermore, terpenoic acid and or triterpenoic acids in the isolatedacidic fraction may be in monomeric form, or in an oligomeric form, suchas a dimer, a trimer, or combinations thereof.

Pharmaceutical Compositions

The composition for use in the invention comprises a therapeuticallyeffective amount of an isolated acidic fraction of mastic gum describedherein, and a pharmaceutically acceptable hydrophobic carrier.

The invention also provides a composition comprising at least onetriterpenoic acid; and a pharmaceutically acceptable carrier. Thetriterpenoic acid may be isolated from a plant product, such as masticgum, as hereinbefore described, or it may be the product of a chemicalsynthesis. Furthermore, the composition may comprise a combination oftriterpenoic acids, some of which are chemically synthesized, and someof which are isolated from one or more plant products. In someembodiments, the composition may consist of at least two triterpenoicacids compounds as the pharmaceutically active ingredients, and apharmaceutically acceptable carrier. In addition, the composition maycomprise dimeric, trimeric and higher oligomeric forms of triterpenoicacids; the oligomers can be formed from both identical and differentmonomeric triterpenoic acids. In some embodiments, the composition mayconsist of at least three triterpenoic acids compounds as thepharmaceutically active ingredients, and a pharmaceutically acceptablecarrier.

For preparation of a composition for therapeutic use, suitable carriersmay be used, such as hydrophobic carriers including pharmaceuticallyacceptable oils, optionally in combination with waxes, as describedherein.

An hydrophobic carrier comprises at least one oil, such as for example amineral oil, a vegetable oil or combinations thereof.

The term “mineral oil” refers to a clear colorless nearly odorless andtasteless liquid obtained from the distillation of petroleum. It mayalso be referred to as white oil, white mineral oil, liquid petrolatum,liquid paraffin or white paraffin oil. In accordance with someembodiments of the invention, the mineral oil is light mineral oil, acommercially available product which may be obtained either as a NF(National Formulary) grade product or as a USP (US Pharmacopoeia) gradeproduct. For use in the invention, the mineral oil is preferably free ofaromatics and unsaturated compounds.

Suitable vegetable oils include, but are not limited to cottonseed oil,olive oil, almond oil, canola oil, coconut oil, corn oil, grape seedoil, peanut oil, saffron oil, sesame oil, soybean oil, or combinationsthereof. In some embodiments, the mineral oil is light mineral oil.

The pharmaceutically acceptable carrier may alternately or in additioncomprise an oil replacement. Oil replacements include alkanes having atleast 10 carbon (e.g., isohexadecane), benzoate esters, aliphaticesters, noncomodogenic esters, volatile silicone compounds (e.g.,cyclomethicone), and volatile silicone substitutes. Examples of benzoateesters include C₁₂C₁₅ alkyl benzoate, isostearyl benzoate, 2-ethyl hexylbenzoate, dipropylene glycol benzoate, octyldodecyl benzoate, stearylbenzoate, and behenyl benzoate. Examples of aliphatic esters includeC₁₂C₁₅ alkyl octonoate and dioctyl maleate. Examples of noncomodogenicesters include isononyl isononanoate, isodecyl isononanoate,diisostearyl dimer dilinoleate, arachidyl propionate, and isotridecylisononanoate. Examples of volatile silicone substitutes include isohexyldecanoate, octyl isononanoate, isononyl octanoate, and diethylene glycoldioctanoate.

Cyclomethicone is an evaporative silicone which may be included in thecarrier to assist in making the composition amenable to ejection from aspray dispenser. Furthermore, due to its evaporative property,cyclomethicone may assist in retaining and fixing the formulation on thesurface to which it is sprayed e.g. a wound site.

The hydrophobic carrier may further comprise at least one wax. Waxesinclude for example, beeswax; vegetable waxes, sugar cane waxes, mineralwaxes, and synthetic waxes. Vegetable waxes include for example,carnauba, candelilla, ouricury and jojoba wax. Mineral waxes include forexample, paraffin wax, lignite wax, microcrystalline waxes andozokerites. Synthetic waxes include for example, polyethylene waxes.

The pharmaceutical composition may be formulated in any of a number offorms such as for example, a capsule (including a softgel capsule), atablet, a gel, a liposome, a suppository, a suspension, an ointment, asolution, an emulsion or microemulsion, a film, a cement, a powder, aglue, an aerosol, a spray and a gel.

For preparing the pharmaceutical composition, the isolated acidicfraction of mastic gum may be suitably formulated as inclusioncomplexes, nanoemulsions, microemulsions, powders and liposomes. In someembodiments, an inclusion complex comprises at least one cyclodextrin.In some embodiments, cyclodextrins comprisehydroxypropyl-3-cyclodextrin. In some embodiments, nanoemulsionscomprise droplets having average particle size of less than 800 nm. Insome embodiments, the droplets have average particle size of less than500 nm. In some embodiments, the droplets have average particle size ofless than 200 nm. In some embodiments, powders are spray dried powders.In some embodiments, liposomes comprise multilamellar vesicles. In someembodiments, a microemulsion comprises a non-ionic surfactant. Non-ionicsurfactants include, without limitation, polyoxyl castor oils,polyoxyethylene sorbitan fatty acid esters (polysorbates), a poloxamer,a vitamin E derivative, polyoxyethylene alkyl ethers, polyoxyethylenesterates, saturated polyglycolyzed glycerides or combinations thereof.

Various formulations of the isolated acidic fraction of mastic gum andpreparation thereof are disclosed herein in Examples 7-11. Thepharmaceutical compositions of the invention may be administered by anymeans that achieve their intended purpose. For example, administrationmay be by, for example, oral, parenteral, topical, transdermal routes,such as, for example, subcutaneous, intravenous, intramuscular,intradermal, intraperitoneal, intraarterial, intrauterine,intraurethral, intracardial, intracerebral, intracerebroventricular,intrarenal, intrahepatic, intratendon, intraosseus, intrathecal, dermal,vaginal, rectal, inhalation, intranasal, ocular, auricular and buccaladministration routes.

The administering may in addition comprise a technique or means such aselectroporation, or sonication in order to assist in their delivery, forexample transdermally. Other techniques which may be employed includefor example, radio frequency or pressurized spray application.

The dosage administered will be dependent upon the age, health, andweight of the subject, the use of concurrent treatment, if any,frequency of treatment, and the nature of the effect desired. The amountof the isolated acidic fraction of mastic gum of the present inventionin any unit dosage form comprises a therapeutically effective amountwhich may vary depending on the recipient subject, route and frequencyof administration.

In general, the amount of the isolated acidic fraction of mastic gumpresent in the pharmaceutical composition may conveniently be in therange from about 0.01% to about 50%, such as, 0.01% to about 25%, suchas 0.01% to about 12%, on a weight per weight basis, based on the totalweight of the composition. For topical use, the percentage of theisolated acidic fraction of mastic gum in the composition may be in therange from about 0.05% to about 2.5%. For administration by injection,the percentage of the isolated acidic fraction of mastic gum in thecomposition may be conveniently in the range from about 0.1% to about7%. For oral administration, the percentage of the isolated acidicfraction of mastic gum in the composition may be in the range from about0.005% to about 7%.

In exemplary embodiments, the amounts of masticadienoic acid andisomasticadienoic acid in compositions consisting of these two compoundsas the active ingredients may be in the range of about 0.05% to about20% for each compound. For administration by injection, the amount foreach may be in the range from about 0.1% to about 10%. For topicaladministration, the amount for each may be in the range from about 0.5%to about 12%. For oral administration, the amount for each may be in therange from about 0.5% to about 15%.

In exemplary embodiments, the amount of oleanonic acid, masticadienoicacid and isomasticadienoic acid in compositions consisting of thesethree compounds as the active ingredients may be in the range of about0.05% to about 15% for each compound. For administration by injection,the amount for each may be in the range from about 0.1% to about 10%.For topical administration, the amount for each may be in the range fromabout 0.5% to about 12%. For oral administration, the amount for eachmay be in the range from about 0.5% to about 15%.

The pharmaceutical compositions of the invention may be manufactured ina manner which is itself known to one skilled in the art, for example,by means of conventional mixing, granulating, dragee-making, softgelencapsulation, dissolving, extracting, or lyophilizing processes.Pharmaceutical compositions for oral use may be obtained by combiningthe active compounds with solid and semi-solid excipients and suitablepreservatives, and/or antioxidants. Optionally, the resulting mixturemay be ground and processed. The resulting mixture of granules may beused, after adding suitable auxiliaries, if necessary, to obtaintablets, softgels, capsules, or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides,e.g., lactose or sucrose, mannitol or sorbitol; cellulose preparationsand/or calcium phosphates, e.g., tricalcium phosphate or calciumhydrogen phosphate; as well as binders, such as starch paste, using,e.g., maize starch, wheat starch, rice starch, potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starchesand also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar,or alginic acid or a salt thereof, such as sodium alginate. Auxiliariesare flow-regulating agents and lubricants, e.g., silica, talc, stearicacid or salts thereof, such as magnesium stearate or calcium stearate,and/or polyethylene glycol. Dragee cores are provided with suitablecoatings which, if desired, are resistant to gastric juices. For thispurpose, concentrated saccharide solutions may be used, which mayoptionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethyleneglycol and/or titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. In order to produce coatings resistant togastric juices, solutions of suitable cellulose preparations, such asacetylcellulose phthalate or hydroxypropymethyl-cellulose phthalate, areused. Dye stuffs or pigments may be added to the tablets or drageecoatings, e.g., for identification or in order to characterizecombinations of active compound doses.

Other pharmaceutical compositions for oral use include push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active compounds in the form of granules, which may be mixedwith fillers, such as lactose; binders, such as starches; and/orlubricants, such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, orliquid paraffin. In addition, stabilizers may be added.

Other pharmaceutical compositions for oral use include a film designedto adhere to the oral mucosa, as disclosed for example in U.S. Pat. Nos.4,713,243; 5,948,430; 6,177,096; 6,284,264; 6,592,887, and 6,709,671.

Pharmaceutical compositions in the form of suppositories consist of acombination of the active compound(s) with a suppository base. Suitablesuppository bases include for example, natural or synthetictriglycerides, polyethylene glycols, or paraffin hydrocarbons.

Formulations for parenteral administration include suspensions andmicroparticle dispersions of the active compounds as appropriate. Insome embodiments, oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, e.g.,sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate,triglycerides, polyethylene glycol-400, cremophor, or cyclodextrins.Injection suspensions may contain substances which increase theviscosity of the suspension include, e.g., sodium carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the suspension may alsocontain stabilizers.

Pharmaceutical compositions can also be prepared using liposomescomprising the active ingredient. As is known in the art, liposomes aregenerally derived from phospholipids or other lipid substances.Liposomes are formed by mono- or multi-lamellar hydrated liquid crystalswhich are dispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolisable lipid capable of forming liposomes can beused. In general, the preferred lipids are phospholipids and thephosphatidyl cholines (lecithins), both natural and synthetic. Methodsto form liposomes are known in the art, as disclosed for example, inPrescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, NewYork, N.Y. (1976) and in U.S. Pat. No. 7,048,943.

Formulations for topical administration include ointments. Suitablecarriers include vegetable or mineral oils, white petrolatum, branchedchain fats or oils, animal fats and waxes. The preferred carriers arethose in which the active ingredient is soluble. Stabilizers, humectantsand antioxidants may also be included, as well as agents imparting coloror fragrance, if desired. Ointments may be formulated for example, bymixing a solution of the active ingredient in a vegetable oil such asalmond oil with warm soft paraffin, and allowing the mixture to cool.

The pharmaceutical composition may comprise an oil-in-water emulsion ormicroemulsion in order to facilitate its formulation for oral,parenteral or topical use Such emulsions/microemulsions generallyinclude lipids, surfactants, optionally humectants, and water. Suitablelipids include those generally know to be useful for creatingoil-in-water emulsions/microemulsions, for example fatty acid glycerideesters. Suitable surfactants include those generally known to be usefulfor creating oil-in-water emulsions/microemulsions wherein lipids areused as the oil component in the emulsion. Non-ionic surfactants may bepreferred, such as for example, ethoxylated castor oil, phospholipids,and block copolymers of ethylene oxide and propylene oxide. Suitablehumectants, if used, include for example propylene glycol orpolyethylene glycol.

The pharmaceutical composition may be formulated in the form of a gel,such as a hydrogel formed from a gel-forming polymer such ascarrageenan, xanthan gum, gum karaya, gum acacia, locust bean gum, guargum. A hydrogel may be combined with an oil-in-water emulsion comprisingthe active ingredient.

The pharmaceutical composition may be formulated in the form of a cementsuch as those comprising polymethylmetacrylate (PMMA) or calciumphosphate, as are used in orthopedic surgery.

The pharmaceutical composition may be formulated in the form of apowder, in particular such as those used for transdermal applicationsusing radio frequency, as described for example, in U.S. Pat. Nos.6,074,688 and 6,319,541 and WO 2006/003659.

The pharmaceutical composition may be formulated in the form of a glue,such as those comprising octocyanoacrylate used for wound closureapplications.

Therapeutic Uses

The present invention provides therapeutic uses and methods of treatingimpaired neurological function, treating skin and scalp disorders,inducing tissue repair and wounds in a subject in need thereof. Themethods comprise administering to the subject a therapeuticallyeffective amount of a composition comprising an isolated acidic fractionof mastic gum, as described herein. In some embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of a composition comprising a combination of at least twotriterpenoic acids selected from: masticadienolic acid;isomasticadienoic acid; isomasticadienolic acid; 3-O-acetylmasticadienolic acid; 3-O-acetyl epimasticadienolic acid; 3-O-acetylisomasticadienolic acid, 3-O-acetyl epi-isomasticadienolic acid; andoleanonic acid. In further embodiments, the method compriseadministering to the subject a therapeutically effective amount of acomposition comprising a combination of at least two triterpenoic acidsselected from masticadienolic acid; isomasticadienoic acid and oleanonicacid.

In some embodiments, the method comprises administering to the subject atherapeutically effective amount of a composition comprisingtriterpenoic acids consisting of masticadienoic acid andisomasticadienoic acid. In further exemplary embodiments, the methodcomprises administering to the subject a therapeutically effectiveamount of a composition comprising triterpenoic acids consisting ofoleanonic acid, masticadienoic acid and isomasticadienoic acid.

The step of administering the compositions may comprise any acceptableroute including oral, topical, parenteral, and transdermal, such as, forexample, parenteral administration includes intravenous, intramuscular,subcutaneous, intradermal, intraperitoneal, intraarterial, intrauterine,intraurethral, intracardial, intracerebral, intracerebroventricular,intrarenal, intrahepatic, intratendon, intraosseus, intrathecal, dermal,vaginal, rectal, inhalation, intranasal, ocular, auricular and buccalroutes of administration.

In some embodiments, the step of administering comprises contactingcells of a particular type, of a particular lineage or at a particularstage of differentiation, with the composition. The cells may be any ofa wide variety of cell types, including in particular, neural cells,neuronal cells, endothelial cells, epithelial cells and stem cells ofsaid lineages. Further, the cells may be of any lineage for example,ectodermal, mesodermal, entodermal lineages and stem cells of saidlineages. In various embodiments, the step of contacting cells iscarried out in vivo, ex vivo or in vitro. In some embodiments, the cellsto be contacted are stem cells and the contacting of the cells iscarried out in vivo, ex vivo and in vitro. In some embodiments, the stemcells are contacted in vivo. In some embodiments, the stem cells arecontacted ex vivo. In some embodiments, the stem cells are contacted invitro.

The method disclosed herein for treating impaired neurological functionis particularly advantageous for subjects afflicted withneurodegenerative conditions and diseases, including in particular,trauma, stroke, vascular dementia, senile dementia, Alzheimer's disease,amyotrophic laterial sclerosis (ALS), multiple sclerosis (MS), andParkinson's disease.

In other cases, the method may be applied in subjects suffering fromimpaired neurological function due to an infection (e.g. viral,bacterial, fungal, parasitic).

In some embodiments the method may be applied in subjects suffering fromimpaired neurological function due to an immunological disorder.

In some embodiments, the impaired neurological function is due toexposure to a drug, such as an anesthetic.

In some embodiments, impaired neurological function may also beassociated with a condition selected from the group consisting ofschizophrenia, bipolar disorder, depression, obesity, anorexia andcachexia.

In some embodiments, skin and scalp disorders include all disorders ofskin, scalp and hair appendages, including for example, nails and hairfollicles. Particular conditions that may benefit from the inventioninclude alopecia, eczema, psoriasis, seborrheic keratosis, seborrhea andskin wounds. Skin wounds include venous leg ulcers, pressure ulcers,diabetic foot ulcers, burns, amputation wounds, decubitus ulcers (bedsore), split-skin donor grafts, skin graft donor sites, medical deviceimplantation sites, bite wounds, frostbite wounds, puncture wounds,shrapnel wounds, dermabrasions, contusions, an infection wounds andsurgical wounds. Wounds may be the result of infection; exposure toionizing radiation; exposure to laser, or exposure to a chemical agent.

In some embodiments, the invention may be effective and economical fortreatment of chronic non-healing wounds. As is known to one of ordinaryskill in the art, the efficacy of a particular treatment in promotingwound healing may be assessed by various criteria, including the rate ofclosure measured by length, width and depth of the wound over time,epithelization rate, formation of granulation tissue and tissue tensilestrength.

In some embodiments, the invention may be particularly effective forinducing and promoting life span extension in humans, non-human mammals,birds and fish.

In some embodiments, the methods disclosed herein for inducing orpromoting tissue regeneration are particularly advantageous for subjectswho have tissue damage, which for example, may be associated with, orthe result of an injury or insult. The methods for inducing or promotingtissue regeneration may be used in subjects who have suffered an injuryor insult selected from the group consisting of a myocardial infarction,a pulmonary embolism, a cerebral infarction, peripheral artery occlusivedisease, a hernia, a splenic infarction, a venous ulcer, an axotomy, aretinal detachment, a wound (for example, a burn wound, bite wound, afrostbite wound, a puncture wound, a shrapnel wound, a contusion, aninfection wound or a surgical wound), an infection and a surgicalprocedure. In some embodiments, the invention may be effective forscar-less repair of wounds.

In some embodiments, the method may be carried out prior to or followingimplantation of a medical device into the subject. Medical devicesinclude, but are not limited to a prosthetic, an artificial organ orcomponent thereof, a valve, a catheter, a tube, a stent, an artificialmembrane, a pacemaker, a sensor, an endoscope, an imaging device, apump, a wire and an implant. Implants include, but are not limited to acardiac implant, a cochlear implant, a corneal implant, a cranialimplant, a dental implant, a maxillofacial implant, an organ implant, anorthopedic implant, a vascular implant, an intraarticular implant and abreast implant.

In some embodiments, the medical device is an organ implant, which mayin certain cases comprise autologous cells of the subject.

In some embodiments, the step of contacting comprises a means selectedfrom the group consisting of electroporation, sonication, radiofrequency, pressurized spray and combinations thereof.

In some embodiments, the step of contacting comprises establishingcontact between interstitial fluid and the composition. This may beparticularly advantageous for wounds which are surrounded byinterstitial fluid. Contact between interstitial fluid and thecomposition may be accomplished by piercing and/or teasing the dermiswith a needle, a microneedle, or an apparatus comprising a plurality ofneedles or microneedles. Such needles or microneedles are preferablynon-hollow and may be fashioned in a plurality for example, on a comb orbrush-like apparatus.

The method of the invention is suitable for application in humans,non-human mammals, fish and birds.

Articles of Manufacture

The method of the invention may encompass use of an article ofmanufacture which incorporates the composition comprising the isolatedacidic fraction of mastic gum described herein.

The pharmaceutical composition may be in the form of a coating on thearticle of manufacture, or may be contained within a vessel which isintegral to the article of manufacture. The pharmaceutical compositionis advantageously present as a coating on devices which are inserted tothe body and are intended for integration therein, for example animplant. The pharmaceutical composition can thus promote tissue closureover the implant due to the activity of the isolated acidic fraction ofmastic gum in inducing cell differentiation.

The pharmaceutical composition may be advantageously incorporated ontoor into articles used in wound healing or tissue repair, for example, adressing or bandage. The pharmaceutical composition can thus promotewound healing due to the activity of the isolated acidic fraction ofmastic gum in inducing cell differentiation.

In other cases, the pharmaceutical composition may be incorporated to adelivery device such as a needle, an injection device or a spraydispenser from which the composition is delivered to a body siterequiring therapy, for example a wound site.

Articles of manufacture include, but are not limited to a fabricarticle, a diaper, a wound dressing, a medical device, a needle, amicroneedle, an injection device and a spray dispenser. In someembodiments, the article of manufacture comprises a plurality ofmicroneedles. Medical devices and implants are as hereinbeforedescribed.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1 Preparation of Isolated Acidic Fraction of Mastic Gum

Mastic resin (10 g) was combined with absolute ethanol (200 ml) and themixture was allowed to stand overnight. The mixture was shaken at 150rpm for ca. 15 minutes, leaving an insoluble gum on the bottom of theflask. Any larger insoluble particles were allowed to settle over 20minutes, and the ethanol was transferred into a new flask. The remainderwas shaken with a fresh portion of absolute ethanol (150 ml) at 200 rpmfor 10 minutes. This ethanol fraction was combined with the firstfraction. The procedure was repeated with another 150 ml portion ofabsolute ethanol which was combined with first two ethanol fractions.Subsequently, the ethanol was removed in vacuo using a rotary evaporator(water-bath temperature 30° C.). Hexane (300 ml) was added to theremaining residue and the mixture was shaken at 150 rpm for two hours.After standing overnight in the closed flask in order to completedissolution of soluble material and precipitation of any insolublematerial, the clear hexane solution was transferred into a cleanpre-weighed flask and the hexane was removed using a rotary evaporator,yielding ca. 6 gram extracted material. The obtained extracted materialwas subsequently dissolved in diethyl ether (300 ML) and extracted witha 5% aqueous sodium carbonate solution (4×100 ML), followed byextraction with 0.1 N aqueous sodium hydroxide (3×100 ML). The two basicaqueous extracts were separately acidified to pH 1-2 by slow addition of10% aqueous hydrochloric acid and subsequently extracted with freshdiethyl ether (3×100 ML). The thus obtained ether fractions werecombined and dried over anhydrous sodium sulfate. After filtering offthe sodium sulfate, the diethyl ether was removed using a rotaryevaporator. This gave ca. 3 gram of isolated acidic fraction of masticgum as a white solid.

For comparison, addition of hexane to the acidic fraction as preparedaccording to the teaching of WO2003/092712 or Parachos et al, (2007),Antimacrobial Agents and Chemotherapy, 51(2), 551, showed that asubstantial amount of that acidic fraction was insoluble in hexane. Whenthis hexane-insoluble material was tested in the in vitro assaydescribed in Example 4 as a 1% ethanolic solution (it was found to belargely insoluble in cottonseed oil), it was found to cause severestress to the cells. Plane ethanol was used as negative control and didnot cause any stress or harm to the cells at the used volumes. Inaddition, a 1% ethanolic solution of the acidic fraction preparedaccording to the current invention induced efficient differentiation ofthe cells into neuronal cells. This result clearly indicates that theacidic fraction of the current invention is free of detrimental materialwhich is present in acidic fractions disclosed in the prior art.

Example 2. Preparation of a 5% (w/w) Composition of Isolated AcidicFraction of Mastic Gum in USP/NF Grade Cottonseed Oil (RPh-Ac)

To 1 gram of the obtained isolated acidic fraction from Example 1 was 19grams of cottonseed oil (USP/NF) was added and the mixture was shaken at150 rpm until a clear and homogeneous composition was obtained (ca.2hours).

Example 3. Isolation and Chemical Characterization of Isolated AcidicFraction of Mastic

Mastic resin from Pistacia lentiscus L. was extracted according toExample 1 to obtain the fraction which was analyzed by reversed phaseHPLC (FIG. 1) in order to identify the major constituents. The HPLCanalysis is consistent with the presence the isolated fraction ofmoronic acid and oleanonic acid in the isolated fraction, on the basisof comparison with analytical standards.

The conditions used for reversed phase HPLC method of the isolatedacidic fraction were: Flow rate: 1 ml/min; detection UV wavelength 220nm; sample concentration 1 mg/ml; injection volume 20 μl; needle andpump back washed with 20% aqueous acetonitrile; ELSD—nitrogen flow 1ml/min; evaporation temperature −80° C.; nebulization temperature 60°C.; 0.8% Acetic acid-ACN gradient.

Gradient Ratio for Acidic Substance and Formulation Samples is Shown inTable 1:

TABLE 1 time A- 0.8% Acetic acid B-ACN 0 20 80 5 20 80 25 7.7 92.3 45 892 48 0 100 55 0 100 57 20 80 67 20 80

Mass spectral data of the isolated acidic fraction (FIGS. 2A-D and FIG.3A) show peaks indicating the presence of monomeric triterpenic acidssuch as moronic acid, oleanonic acids and others (MH⁺ at 455 Da; M⁺+Naat 477 Da. Also present in the mass spectral data are peakscorresponding to dimeric triterpenoic acids (MH⁺ at 910 Da; M⁺+Na at 932Da), as well as peaks indicative of trimeric forms of triterpenoic acids(MH+ at 1364 Da; M⁺+Na at 1387).

In order to isolate and determine the structure of further mainconstituents of the isolated acidic fraction, a preparative HPLC methodwas developed. Using this method, six major constituents of the isolatedacidic fraction were subsequently isolated by preparative HPLC.

A preparative HPLC method was developed on a 30×250 mm preparativecolumn (ACE-121-2530). Samples of the isolated fraction (ca. 75 mg perrun) were injected using a 5 ML loop.

Method Details:

Detection wavelength: 205 nm; Flowrate: 15 ml/min

Eluents:

-   -   1. 0.8% acetic acid:acetonitrile:THF=25:72:3    -   2. 0.8% acetic acid:acetonitrile:THF=15:82:3    -   3. 0.8% acetic acid:acetonitrile:THF=10:87:3    -   4. 0.8% acetic acid:acetonitrile:THF=5:92:3        Schedule: Prior to conditioning of column, it has to be washed        with Acetonitrile HPLC grade during at least 40 minutes. The        total run time is ca. 155 minutes.        Conditioning: Run Eluent 1 for 30 mins.        Load: Inject 5 ML solution of 75 mg sample in MeOH        Elution:        Eluent 1: 0-10 minutes        Eluent 2: up to elution of peak 2        Eluent 3: until 10 minutes after elution of peak 4        Eluent 4: until 10 minutes after elution of peak 6

The chromatogram of the preparative HPLC method is shown in FIG. 4.

The compounds corresponding to the six indicated peaks in FIG. 4 wereisolated and characterized by ¹H-NMR and ¹³C-NMR.

Peak 1 and peak 2 were indeed shown to be respectively moronic acid andoleanonic acid upon comparison of the NMR spectra with literature data.The ¹H-NMR and ¹³C-NMR spectra of these two acids are shown in FIGS.5A-B (Peak 1, moronic acid) and 6A-B (Peak 2, oleanonic acid),respectively. Moronic acid was also positively identified by comparisonwith a commercial sample. Oleanonic acid was further compared and foundto be identical with, a sample prepared by oxidation of oleanolic acidaccording to a literature method (Helv. Chim. Acta Vol. 83, p. 1766(2000).

Peaks 3-6 were shown to be masticadienoic acid (peak 3),isomasticadienoic acid (peak 4), 3-OAcetyl-masticadienolic acid (peak 5)and 3-O-acetyl-isomasticadienolic acid (peak 6), respectively. The¹H-NMR and ¹³C-NMR spectra of these is shown in FIGS. 7A-B, FIGS. 8A-B,FIGS. 9A-B and FIGS. 10A-B, respectively. (For reference—Parachos et al,(2007), Antimacrobial Agents and Chemotherapy, 51(2), 551 and referencestherein.)

Example 4. Biological Studies of RPh-Ac in Retinal Pigment Epithelium(RPE) Cells

Studies aimed at evaluating effects of RPh-Ac on various cell lines ofhuman origin led to use of ARPE-19 cells, a non-malignant human retinalpigment epithelial cell line.

The retinal pigment epithelium (RPE) is a single layer of hexagonalpigmented epithelial cells of neuronal origin, which forms the outermostcell layer of the eye retina and is attached to the underlying choroid.RPE functions include support, nourishment and protection of theunderlying photoreceptors of the neuro-retina.

ARPE-19 cells are involved in the phagocytosis of the outer segment ofphotoreceptor cells, in the vitamin A cycle, where they isomerizeall-trans retinol to 11-cis retinal and in supplying the photoreceptorswith D-glucose, amino acids and ascorbic acid.

Although in vivo the RPE is pigmented, ARPE-19 cells do not form melaninand are not pigmented. In culture the cells grow as spindle shaped andas polygonal cells.

Methods

ARPE-19 cells (obtained from the American Type Culture Collection,(ATCC)) were plated in flat bottom 96 well tissue culture microplates(Costar) at a concentration of 2-5×10³ cells per well (1-2.5×10⁴cells/mL) in a growth medium consisting of DMEM:Ham F-12, 1:1,supplemented with 10% Fetal Bovine Serum, 200 mM glutamine, 100 units/mLpenicillin and 100 μg/mL streptomycin. The cells were allowed to adhereto the plate surfaces overnight prior to treatment with RPh-Ac.

RPh-Ac was prepared essentially as described in Example 2, to provide a2.5% solution in a carrier composed of cottonseed oil. The preparationswere added to the wells at volumes of 0.5 μl, 1 μl, 1.5 μl, 2 μl. Thesevolumes, introduced into an overall sample medium volume of 200 μl,correspond to final RPh-Ac concentrations of 0.0125%, 0.025%, 0.0375%and 0.05%, respectively. The oil carrier served as a vehicle control andwas applied to control cultures at the same volumes.

The cultures were incubated in a 37° C., 5% CO₂ incubator for 48 hrs.The medium was then removed, the cultures washed twice with phosphatebuffered saline (PBS), fixed with absolute methanol for 10 min andstained with Hemacolor® reagents (Boehringer Mannheim), which staincells in a manner similar to Giemsa, and may be used in a quantitativecell viability assay (Keisari, Y. A colorimetric microtiter assay forthe quantitation of cytokine activity on adherent cells in tissueculture. J. Immunol. Methods 146, 155-161, 1992).

To determine the expression of marker proteins, Synaptophysin,phosphorylated Akt (pAkt^(Ser473)) and phosphorylated GSK3-beta^(Ser9))in response to treatment of the cells with RPh-Ac, cells were plated onsterile glass coverslips immersed in 24 well microplates at aconcentration of 5-10⁴ cells/well in a medium consisting of 1:1 mixtureof Dulbecco's minimal essential medium (DMEM) and Ham F12 medium,supplemented with 10 fetal bovine serum and penicillin (100 units/ml),streptomycin (100 μg/ml) and glutamine (2 mM).

The cells were allowed to adhere overnight to cover slips and 2.5%RPh-Ac in cottonseed oil was added to the culture at a volume of 20μl/ml medium and incubated in a 37° C., 5% CO₂ for 48 hrs. The oilcarrier served as a vehicle control and was applied to control culturesat the same volume.

The cells were then washed 2× with PBS and fixed with 4%paraformaldehyde. To determine protein expression of, Synaptophysin,pAkt^(Ser473), and phosph-GSK3-beta^(Ser9) in the cells, the glasscoverslips were stained with mouse/rabbit monoclonal antibodies (Ab.)direct against human Synaptophysin, pAkt^(Ser473), andpGSK3-beta^(Ser9), followed by secondary FITC-labeled anti-mouse/rabbitIgG. The cells nuclei were counter stained with DAPI. Test and controlpreparation were then evaluated in a fluorescence microscope. Theresults are presented in FIGS. 13A-C, herein below.

Results

Treatment of ARPE-19 RPE cells with RPh-Ac was unexpectedly found toinduce dramatic morphological changes that are unequivocallycharacteristic of neuro-differentiation. The morphological changes didnot occur in control cultures treated with oil carrier alone, andsimilar results were seen among the test cultures treated with RPh-Ac,regardless of the oil used as the carrier for the active compound. Themorphological changes were also associated with cessation in cellproliferation, further supporting the conclusion that RPh-Ac inducesneuro-differentiation.

Control oil-treated cultures displayed the typical spindle shaped andpolygonal growth pattern characteristic of ARPE-19 RPE cells (FIG. 11A).After 48 hours of incubation in culture, cells treated with RPh-Ac(0.01%; 0.2 mg/ml) were altered in shape, and developed thick, denselystaining very long single protrusions reminiscent of neuronal cellaxons. After 48 hour of incubation, cells treated with RPh-Ac (0.025%;0.25 mg/ml) displayed a larger number of thinner long protrusionsreminiscent of dendrites; and after 48 hours of incubation with RPh-Acthe thin long protrusions formed junctions with similar protrusions inadjacent cells creating a network of inter-connected cells, potentiallycapable of communicating information between one another (FIG. 11B).Similar networks occur normally between neurons in the central nervoussystem and enable transmission and processing of information.

While control cells proliferated during the 48 hour incubation period(FIG. 11C), RPh-Ac treated cells rapidly ceased to proliferate and thecells remained in sparse density, further supporting the notion of celldifferentiation.

A Scoring System for the Potency in Inducing Cell Differentiation

On the basis of the above results, a scoring system was developed toevaluate the potency of the fractions for inducing differentiation incell culture, with cells plated 2×10³ per well. The grades and theirrespective descriptions are set out in Table 2.

TABLE 2 Effect Grade Proliferation rate High = 0 Medium = 1 Low = 2Cells are forming elongated protrusions No = 0 protrusions = 1 neuronlike = 2 Neurites (neuron-like elongations)/ ≤2 = 0 body ratio >2 ≤ 3 =1 >3 = 4 Percent of differentiated cells ≤10% = 0 >10% ≤ 30% = 1 >30% ≤70% = 2 ≥70% = 3 Clearly visible junctions between ≤30%, =0 neuritesand/or cell bodies >30% < 70%, =1 ≥70% = 2 Visible, clear synaptic-likebuttons along <30% = 0 the neurites and at the ends of the >30% < 50% =1 neurites ≥70% = 2

The same assays were performed using the following treatments:

-   -   Cottonseed oil vehicle (negative control) (results shown in FIG.        11C)    -   Acidic fraction according to WO2003/097212 and Hexane insoluble        material isolated from acidic fraction according to the teaching        of WO 2003/097212, 1% in ethanol (Results shown in FIG. 12A)    -   RPh-Ac, 1% in ethanol (Results shown in FIG. 12B)    -   Ethanol vehicle (negative control) (Results shown in FIG. 12C).

Results according to the above scoring table for evaluating thedifferentiation observed in FIG. 11 and FIG. 12, in response to thevarious treatments, is depicted in Tables 3 and Table 4, below.

TABLE 3 Name of fractions Acidic WO Vehicle 2003/092712 RPh-Ac(cottonseed oil) Volume (ul) 3.5 5 7 10   2 3.5 5 7 2 3.5 5 7Differentia- 1 2 2 4** 4 3 10 —* 0 0 0 0 tion grade Differentia- 1 2 23** 3 3 4 —* 0 0 0 0 tion score *In these amounts the cells had reachedfull differentiation and subsequently died naturally before the end ofthe 48 hours incubation. **Cells were clearly stressed and showed atbest only hints of differentiation.

TABLE 4 Name of fractions Hexane insoluble Acidic WO2003/092712 RPh-AcVehicle (1% w/w in) ethanol (1% w/w in ethanol) (Ethanol) Volume (ul)3.5 5  7  10   2 3.5 5 7 2 3.5 5 7 Differentiation grade 1 1** 2** 2** 43 15 —* 0 0 0 0 Differentiation score 1 1** 2** 2** 3 3 5 —* 0 0 0 0 *Inthese amounts the cells had reached full differentiation andsubsequently died naturally before the end of the 48 hours incubation.**Cells were clearly stressed and showed only early hints ofdifferentiation.

The cells were then washed 2× with PBS and fixed with 4%paraformaldehyde. To determine protein expression of, Synaptophysin,pAkt^(Ser473), and phosph-GSK3-beta^(Ser9) in the cells, the glasscoverslips were stained with mouse/rabbit monoclonal antibodies directagainst human Synaptophysin, pAkt^(Ser473), andphosphor-GSK3-beta^(Ser9), followed by secondary FITC-labeledanti-mouse/rabbit IgG. The cells nuclei were counter stained with DAPI.Test and control preparation were then evaluated in a fluorescencemicroscope. The results are presented in FIGS. 13A-C, herein below.

The results shown in FIG. 13A, demonstrate that in cells treated withthe composition RPh-Ac, high levels of Synaptophysin clusters wereevident in cellular processes (Top panel), as compared to control cells(bottom panel) where Synaptophysin is not expressed at all.Synaptophysin is an abundant synaptic vesicle in the brain and regulatesactivity-dependent synapse formation in neurons. Synaptophysin has beenwidely used to identify synapse formation in differentiating neuronalcells.

The activities of GSK-3 are negatively regulated by serinephosphorylation. Inactivation of GSK-3beta is a principal regulatorytarget of the phosphatidyl-inositol 3-kinase/Akt survival pathway andexert some of its neuroprotective effects. GSK-3 inactivation has beenproposed as a mechanism to promote neuronal survival functions,including regulation of neurite formation and modulation ofglutamatergic neurotransmission. GSK-3 beta inhibition has also beenimplicated in axonal morphology and synaptic protein clustering indeveloping neurons. Dysregulation of GSK-3-mediated substratephosphorylation and signaling pathways has been implicated in thepathophysiological conditions of a variety of diseases, includingAlzheimer disease, type 2 diabetes, and cancer schizophrenia and mooddisorders.

The results shown in FIG. 13B, demonstrate that cells treated with thecomposition RPh-Ac exhibit high levels of pAkt^(Ser473) (Top panel), ascompared to vehicle treated cells (bottom panel). The results shown inFIG. 13C, demonstrate that cells treated with the composition RPh-Acexhibit high levels of pGSK-beta^(Ser9) as well as changes in itslocalization, as compared to vehicle treated cells (bottom panel). Theseresults suggest that composition RPh-Ac inactivates GSK3 beta by itsphosphorylation on Ser9 through activation of Akt (phosphorylation ofSer 473).

Example 5. Synergistic Effect of Combinations of Specific TriterpenodisCompounds of the Isolated Acidic Fraction in Rat MCAO Model

Stroke is a prominent cause of serious, long-term disability and thethird leading cause of death in the United States. Ischemic strokescomprise over 88% of all strokes, making them the most common type ofcerebrovascular injury. Ischemic conditions in the brain cause neuronaldeath, leading to permanent sensorimotor and cognitive deficits. TheMiddle Cerebral Artery occlusion (MCAO) model is a reliable model forstroke in rats and mimics the human condition. Occlusion of MCA leads toinjury of the sensorimotor cortex due to neuronal loss. The level ofthis injury can be assessed by histology evaluation of the infarct sizeand various behavior tests. Behavioral improvement after strokeinduction and low infarct volumes indicate a better pathologicalcondition and are probably the result of either neuroprotection orneurogenesis. Therefore, the MCAO model can serve as reliable testingsystem to evaluate efficacy of drugs as a therapeutic agents forimpaired neurological conditions, such as, for example, stroke andneurodegenerative diseases/conditions.

For the experiments described herein, the animals are anesthetized usingketamine/xylazine solution. The animal's neck is then shaved and amidline incision is made in the skin of the neck, and the tissueunderneath is bluntly dissected. The right common carotid artery (CCA)and its junction with the external carotid artery (ECA) and internalcarotid artery (ICA) are exposed by blunt dissection. The CCA is thentransiently closed by positioning around it a 3-0 silk suture material.The ECA is permanently occluded with the same type of suture material. Athird closure, transient, is also done in the ICA with 3-0 silk suturematerial.

A small hole is cut in the ECA and the nylon thread is inserted into theICA while avoiding entrance into the pterygopalatine artery. The threadis inserted 21-25 mm until a slight resistance is felt. Then a 3-0 silksuture knot secures the thread.

Following surgery, the rats are returned to their cages and remain undera heating lamp until they awake.

Two parameters are monitored and recorded during the ischemic surgeryprocedure: animal's core temperature and blood glucose level (BGL).

Core temperature is monitored using rectal probe (YSI USA model 400)connected to a measuring unit (Cole Parmer model 8402-00) during theentire surgery period. Ischemic insult is started when rectaltemperature is 37-38° C.

In view of the beneficial effect of RPh-Ac fraction composition on cells(as exemplified above herein), various compounds comprised within theRPh-Ac fraction were tested for their in-vivo effects.

Abbreviations used for the tested compounds and combinations:

OA: Oleanonic acid

MDA: Masticadienoic acid

IMDA: Isomasticadienoic acid

The rats of the different groups were injected twice a week subcutaneouswith 25 microliters of designated test item. First injection was giventhree hours after stroke induction.

The following formulations were used as test items for injection:

-   -   Vehicle: cottonseed oil    -   IMDA: 0.65% (w/w) in cottonseed oil    -   MDA: 0.65% (w/w) in cottonseed oil    -   IMDA and MDA: each 0.65% (w/w) in cottonseed oil    -   IMDA, MDA and OA: each 0.65% (w/w) in cottonseed oil        MDA alone; IMDA alone; a combination of MDA+IMDA; and a        combination of OA+MDA+IMDA, were tested using a rat MCAO model,        for their therapeutic capacity. Stroke induced rats were treated        with each of the tested compounds and the improvement of their        sensorimotor abilities were tested using the adhesive removal        test. In this test, a small adhesive tape is attached to the        front paws of the rats and serves as a tactile stimulus. The        time to remove the tape is recorded. The adhesive removal tape        was conducted before the induction of stroke, to evaluate the        base line time of each group and at 27 days after the injury,        following twice a week injections of the test items.        Results:

As shown in the graphs of FIG. 14A, which show the time (in seconds) ittook the rats to remove the adhesive tape from the contralateral paw,IMDA alone, was able to improve the score of treated rats in theadhesive removal test, compared to vehicle treated rats. MDA alone wasonly slightly better than the vehicle control. However, the combinationof IMDA+MDA or OA+MDA+IMDA (right thick diagonal line), clearly improvedthe efficacy of the treatment and the animals were back to their baseline (day 0, DO) performance on day 27 (D27).

Since IMDA by itself almost restored the base line score of the rats,the ratio of the beneficial test items relative to the base line wasanalyzed. As shown in FIG. 14B, IMDA, which significantly improved therat's sensorimotor condition, was still 10 fold worse than the base linescore. This suggests that IMDA by itself is limited in its effect andcannot fully restore the base line sensorimotor function. However,surprisingly, the combinations of IMDA and MDA or the combination ofIMDA, MDA and OA further increased efficacy, resulting in dramaticimprovement of the sensory-motor capacity to a level even lower than therecorded baseline. These results demonstrate clearly the strongsynergistic effect of the combinations of IMDA, MDA and OA.

The results presented herein strongly support the therapeutic potentialof combination of isomaticadienoic acid and masticadienoic acid or thecombination of isomasticadienoic acid, masticadienoic acid and oleanonicacid, and indicate a strong synergistic effect of these compounds oneach other with respect to treatment of impaired neurological functionand regeneration of neuronal tissue.

Example 6. Wound Healing in Rats

For the rats used in the MCAo model of Example 5, the healing of thesurgical wounds was used as an indicator for the wound healing potentialof the tested formulations used in Example 5.

The pictograms shown in FIG. 15 were taken of wounds during day 7 of theMCAO model, for rats treated with vehicle (plain cottonseed oil, LeftPanel), RPh-Ac (middle panel) and the mixture of oleanonic acid,masticadienoic acid and isomasticadienoic acid (right). The photographsclearly indicate that the wounds of animals treated with RPh-Ac (middlepanel) or with the mixture of oleanonic acid, masticadienoic acid andisomasticadienoic acid (right panel) are in a more advanced stage ofhealing in comparison with the wound of the vehicle treated animal.

Example 7. Preparation of Complexes of Cyclodextrin

Cyclodextrins, by virtue of their ability to form inclusion complexeswith many drugs, can substantially increase the aqueous solubility ofbiopharmaceuticals, in particular those that are defined aswater-insoluble such as particular terpenoid compounds. Cyclodextrinsare water-soluble compounds, which can form reversible complexes withpoorly water-soluble molecules resulting in a soluble molecularinclusion complex. When the inclusion complex of the drug-cyclodextrincombination is diluted in a sufficiently large volume of water or blood,it dissociates rapidly, releasing the sequestered pharmacologicallyactive agent.

Complexation of isolated acidic fractions described herein with β-HPCDwill be performed as follows:

a. Dissolution of pre weighed gum mastic fraction in a minimum amount ofnon-polar solvent such as hexane, heptane, or the like.

b. Dropwise addition of the non-polar solvent to the β-HPCD powder.

c. Drying at 50-80° C. until non-polar solvent evaporates.

d. Mixing with necessary amount of water.

e. Dissolution with sonication and heating.

f. Filtration through 0.2-0.45 μm filter.

Example 8. Preparation of Nanoemulsions of Isolated Acidic Fraction ofMastic Gum

Liquid oil-in-water nanoemulsion formulations are to be prepared by highpressure emulsification techniques of all lipid ingredients and theactive component dissolved in the lipid oil phase and emulsified with anaqueous phase, projected to result in the formation of stable, sphericand uniformly dispersed drug-containing lipid nanodroplets. The emulsiondroplet size reduction is essential to generate drug formulations withhigh stability. Preferred nanoemulsion droplets have a mean droplet sizeof less than one micron (generally in the range of 0.1-0.2 μm) uniformlydispersed in an aqueous phase. The uniqueness of the large internalhydrophobic oil core of the nanoemulsion droplets provides highsolubilization capacity for water insoluble compounds.

1. Preparation of Oil Phase

The oil phase is composed of 13% lipoid E-75, 0.026% αTP-succinate,propylparaben as antioxidant and 86.9% Miglyolo 810. Gum mastic fractionprepared as in Example 1 is dissolved in the oil phase. The componentsare mixed with mild heating until a homogenous completely solubilizedsolution is obtained.

2. Preparation of Aqueous Phase

The aqueous phase is composed of 0.1% EDTA, 0.5% Tween-80, 2.3%glycerol, methylparaben as preservative and 97.1% water. pH was adjustedto 7.4 by NaOH 1N.

3. Mixing of Oil and Aqueous Phases

Oil phase (3.7 g) is heated and added to 70 ml of the aqueous phase(preheated). The mixture is gently stirred for 10-15 min at roomtemperature.

4. Preparation of Oil-in-Water Coarse Emulsion

An oil-in-water emulsion is prepared using the medium size dispenser andhigh shear homogenizing unit Polytronn, at 20,000 rpm for 5 min.

5. Sizing the Emulsion to Submicron Range by Gaulin® High PressureHomogenizer

The droplet size of the emulsion obtained after step 4 is reduced to thesubmicron (nanosize) range by submitting the emulsion to high shearhomogenization using the Gaulinm Microlab 70 high pressure homogenizerat 800 bar pressure. A total of 5-6 cycles should be performed to obtainhomogenous nanoemulsion droplets having average particle size of lessthan 200 nm. Particle size is to be determined by photon correlationspectroscopy (PCS) using a N4MD particle size analyzer (Coulter®Electronics, UK). When most of the particles (>90%) are smaller than 200nm, the sizing process is determined to be complete.

6. Sterile Filtration

Filtration at aseptic conditions of the nanoemulsion to sterile vialsusing a 0.2 μm PES sterile filter and storage at 40° C.

Example 9. Preparation of Spray-Dried Powder

A convenient process for manufacturing the gum mastic fraction-lipidmixture product is by direct spray-drying of the formulation from amixture of non-polar solvent dispersion containing all the lipidingredients and water containing the hydrophilic components, taking intoaccount cost effectiveness and upscaling considerations. The selectedspray-drying method is optimized in order to get a fine, free-flowingpowder. The gum mastic fraction is to be dissolved in the lipid phasecontaining the lipid ingredients lecithin, tricaprin (capric acidtriglyceride), tocopherol succinate and warmed (−40° C.) in a non-polarsolvent until a good dispersion is obtained. A dispersion of fumedsilicon dioxide (Cab-O-Sil®) in water (5%) will be prepared by swellingthe powder in purified water. The resultant slurry (prewarmed to 40° C.)may then be poured slowly into the non-polar solvent lipid dispersionand the mixture agitated at 40° C. for about 1 hr until a homogenousdispersion is obtained. The mixture is then to be spray-dried using theYamato Pulvis® GA32 spray-dryer. Typical spray-drying conditions are:flow rate 7 ml/min, inlet temperature 130° C., outlet temperature 70°C., and drying air flow 0.5 m³/min. A homogeneous dry powder containingthe gum mastic fraction-lipid mixture is expected to be obtained.

The gum mastic fraction—lipid mixture formulation prepared by the directspray drying process is expected to show good water dispersibility, thusbeing suitable for the preparation of solid-dosage forms such as hardgelatin capsules or tablets for enhanced oral delivery with good oralbioavailability.

Example 10. Preparation of Liposomal Preparations

Lipids containing dissolved isolated acidic fraction of mastic gum areto be dissolved in 100 ml dichloromethane in a round bottom flask, andstirred for 30 min at room temperature until a clear transparentsolution is obtained. Solvent will be evaporated using a rotaryevaporation unit at 39° C. Typical conditions include rotation of theflask at 4.5 rpm, 5 min under atmospheric pressure, followed by 10-30min (until full evaporation of the solvent) under weak vacuum, andfinally 15 min under full vacuum. At the end of the evaporation processa uniform lipid film will be created. The lipid film will be dissolvedin 15 ml isotonic buffer. Liposomes are prepared by vigorous shaking for10-30 min using multi-wrist shaker, until a uniform and milky dispersionof multilamellar vehicle (MLV) is formed and no remaining lipid filmremains apparent. In order to obtain an equilibrated and homogenousliposome preparation, the flask may be further shaken at 37° C. for30-90 min. at 270 rpm.

Example 11. Preparation of Microemulsions Containing Isolated AcidicFraction of Mastic Gum

Several surfactants commonly used in parenterals may be utilized todevelop water-in-oil and oil-in-water-microemulsions acceptable forinjectable, oral and topical use. Pharmaceutically acceptablesurfactants suitable for the formation of microemulsion formulationsinclude non-ionic surfactants including polyoxyl 40 hydrogenated castoroil (sold under the trade name Cremophor RH40®), polyoxyl 35 castor oil(sold under the trade name Cremophor® EL), polyoxyethylene sorbitanfatty acid esters (polysorbates), poloxamers (Pluronics®), vitaminE-TPGS 1,000 (VE-TPGS 1,000), polyoxyethylene alkyl ethers, Solutol®HS-15, Tagat® TO, Peglicol 6-oleate, polyoxyethylene sterates, orsaturated polyglycolyzed glycerides, all of which are commerciallyavailable. The preferred surfactants include polyoxyl 40 hydrogenatedcastor oil (Cremophor® RH40®), polyoxyl 35 hydrogenated castor oil(Cremophor® EL), polyoxyethylene sorbitan fatty acid esters(polysorbates), poloxamers (Pluronics®), and vitamin E-TPGS 1,000. Thetotal amount of the surfactant present in the composition will begenerally from about 100 to about 700 mg/g, and preferably from about300 to about 500 mg/g.

Preparation of microemulsions containing the isolated acidic fractionmay be performed by dissolving the isolated acidic fraction in anappropriate amount of oil such as medium chain tryglycerides (Miglyol)in a suitable vial. The vial is then capped. The vial is put into awater bath of about 50-60° C. and shaken gently until all of the drugmaterial is completely dissolved. After the vial is cooled to roomtemperature, an appropriate amount of surfactant (such as Cremophor® ELor VE-TPGS) is added and followed by the mixture of mono- anddi-glycerides of fatty acids, if any. The vial is then capped and placedinto the water bath of about 50-60° C. The vial is shaken gently toobtain a clear, uniform solution. This solution can be filled into HPMCcapsules and stored at room temperature before oral dosing.Alternatively, the substituted polymer powders (such as HPMC) can beadded into the solution with adequate agitation (i.e., stirring,shaking) to obtain a uniform polymer suspension. The resultingcomposition can then be filled into either soft gelatin or hard gelatincapsules and stored at room temperature before oral dosing.Alternatively the microemulsion formulation can be used as a topicallyor filtered through 0.2 um membranes to be administered parenterally.

The microemulsions have good water-dispersibility properties andself-emulsify when diluted in aqueous media to form small nanometricmicelles that with enhanced bioavailability.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

The invention claimed is:
 1. A process for preparing an acidic fractionof mastic gum, the process comprising the steps of: (a1) treating masticgum with a polar organic solvent; (b1) isolating a fraction soluble insaid polar organic solvent; (c1) optionally removing said polar organicsolvent; (d1) treating the soluble fraction obtained in step (b1) or(c1) with a non-polar organic solvent, (e1) isolating a fraction solublein said non-polar organic solvent; (f1) optionally removing saidnon-polar organic solvent; or (a2) treating mastic gum with a non-polarorganic solvent; (b2) isolating a fraction soluble in said non-polarorganic solvent; (c2) optionally removing said non-polar organicsolvent; (d2) treating the soluble fraction obtained in step (b2) or(c2) with a polar organic solvent, (e2) isolating a fraction soluble insaid polar organic solvent; (f2) optionally removing said polar organicsolvent; and further comprising (g) dissolving the fraction obtained instep (e1), (e2), (f1) or (f2) in an organic solvent; (h) treating thesolution obtained in step (g) with a basic aqueous solution so as toobtain a basic aqueous fraction; and (i) acidifying the basic aqueousfraction obtained in step (h) with an acid solution so as to obtain saidacidic fraction of mastic gum.
 2. The process according to claim 1,further comprising the steps of: (j) extracting the acidified aqueousfraction obtained in step (i) with an organic solvent; (k) optionallycontacting the organic fraction obtained in step (j) with a drying agentso as to remove remaining water; (l) removing organic solvent and/orexcess acid from the fraction obtained in any of steps (i), (j) or (k);and (m) dissolving the isolated fraction obtained in step (l) in apharmaceutically acceptable carrier.
 3. The process according to claim1, comprising: (a1) treating mastic gum with a polar organic solvent;(b1) isolating a fraction soluble in said polar organic solvent; (c1)optionally removing said polar organic solvent; (d1) treating thesoluble fraction obtained in step (b1) or (c1) with a non-polar organicsolvent, (e1) isolating a fraction soluble in said non-polar organicsolvent; (f1) optionally removing said non-polar organic solvent; (g)dissolving the fraction obtained in step (e1) or (f1) in an organicsolvent; (h) treating the solution obtained in step (g) with a basicaqueous solution so as to obtain a basic aqueous fraction; and (i)acidifying the basic aqueous fraction obtained in step (h) with an acidsolution so as to obtain said acidic fraction of mastic gum.
 4. Theprocess according to claim 1, wherein steps (a1) to (c1), steps (a2) to(c2), steps (d1) to (f1) and/or steps (d2) to (f2) are repeated for aplurality of cycles.
 5. The process according to claim 2, wherein theorganic solvent in step (g) and in step (j) is independently selectedfrom the group consisting of dialkyl ethers, alkyl-aryl ethers, diarylethers, ketones, halogenated hydrocarbons, C₅-C₁₄ aromatic hydrocarbons,C₅-C₁₄ perfluoroalkanes and a combination thereof.
 6. The processaccording to claim 2, wherein the polar organic solvent comprisesethanol, the non-polar organic solvent comprises hexane and the organicsolvent of step (j) comprises diethyl ether.
 7. The process according toclaim 2, wherein the polar organic solvent is ethanol, the non-polarorganic solvent is n-hexane and the organic solvent of step (j) isdiethyl ether.
 8. The process according to claim 1, wherein the basicaqueous solution in step (h) is prepared from an inorganic base selectedfrom the group consisting of sodium carbonate, sodium hydroxide,potassium carbonate, potassium hydroxide, ammonium hydroxide, sodiumbicarbonate, sodium phosphate, lithium hydroxide, lithium carbonate, andpotassium phosphate.
 9. The process according to claim 8, wherein theinorganic base is sodium carbonate having concentration in the rangefrom 2 to 20% w/w in water.
 10. The process according to claim 1,wherein the mastic gum is obtained from a species of Pistacia selectedfrom the group consisting of P. lentiscus, P. atlantica, P. palestina,P. saportae, P. terebinthus, P. vera and P. integerrima.
 11. The processaccording to claim 2, wherein the carrier is a hydrophobic carrierselected from the group consisting of at least one oil, at least one waxand combinations thereof.
 12. The process according to claim 11, whereinthe at least one oil is selected from the group consisting of cottonseedoil, almond oil, canola oil, coconut oil, corn oil, grape seed oil,olive oil peanut oil, saffron oil, sesame oil, soybean oil andcombinations thereof.
 13. The process according to claim 1, wherein thepolar organic solvent is selected from an alcohol, an ether, an ester,an amide, an aldehyde, a ketone, a nitrile, and combinations thereof.14. The process according to claim 1, wherein the polar organic solventis selected from the group consisting of methanol, ethanol, propanol,isopropanol, 1-butanol, 2-butanol, sec-butanol, t-butanol, 1-pentanol,2-pentanol, 3-pentanol, neopentanol, 3-methyl-1-butanol,2-methyl-1-butanol, 3-methyl-2-butanol, 2-methyl-2-butanol,ethyleneglycol, ethyleneglycol monomethyl ether, diethyl ether,methylethyl ether, ethylpropyl ether, methylpropyl ether,1,2-dimethoxyethane, tetrahydrofuran, dihydrofuran, furan, pyran,dihydropyran, tetrahydropyran, methyl acetate, ethyl acetate, propylacetate, acetaldehyde, methylformate, ethylformate, ethyl propionate,methyl propionate, dichloromethane, chloroform, dimethylformamide,acetamide, dimethylacetamide, N-methylpyrrolidone, acetone, ethylmethylketone, diethyl ketone, acetonitrile, propionitrile, and a combinationthereof.
 15. The process according to claim 1, wherein the non-polarorganic solvent is selected from the group consisting of acyclic orcyclic, saturated or unsaturated aliphatic hydrocarbons and aromatichydrocarbons, each of which is optionally substituted by one or morehalogens, and a combination thereof.
 16. The process according to claim1, wherein the non-polar organic solvent is selected from the groupconsisting of C₅-C₁₀ alkanes, C₅-C₁₀ cycloalkanes, C₆-C₁₄ aromatichydrocarbons and C₇-C₁₄ perfluoroalkanes, and a combination thereof. 17.The process according to claim 1, wherein the non-polar organic solventis selected from the group consisting of pentanes, hexanes, heptanes,octanes, nonanes, decanes, cyclopentane, cyclohexane, cycloheptane,benzene, toluene, xylene, and isomers and mixtures thereof.
 18. Theprocess according to claim 1, wherein, the acid solution of step (i) isan aqueous solution of hydrochloric acid or phosphoric acid.
 19. Theprocess according to claim 3, further comprising the steps of: (j)extracting the acidified aqueous fraction obtained in step (i) with anorganic solvent; (k) optionally contacting the organic fraction obtainedin step (j) with a drying agent so as to remove remaining water; (l)removing organic solvent and/or excess acid from the fraction obtainedin any of steps (i), (j) or (k); and (m) dissolving the isolatedfraction obtained in step (l) in a pharmaceutically acceptable carrier.